Novel bone mineralization protein expression systems, and methods of studying their intracellular signaling pathways

ABSTRACT

The present invention is directed to methods of switching a differentiation of a cell from a non-osteogenic lineage into an osteogenic lineage. The present invention is also directed to methods of generating a model system for assessing the intracellular signaling pathways of bone growth factors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 11/433,059 filed on May 12, 2006, which is acontinuation of U.S. application Ser. No. 09/959,578, filed Jun. 21,2002, which claims the benefit of U.S. Provisional Application No.60/132,021, filed on Apr. 30, 1997.

FIELD OF THE INVENTION

The field of the invention relates generally to osteogenic cells and theformation of bone and boney tissue in mammalian species. Specifically,the invention concerns a novel family of proteins, and nucleic acidsencoding those proteins, that enhances the efficacy of bonemineralization in vitro and in vivo. The invention provides methods fortreating a variety of pathological conditions associated bone and boneytissue, such as, for example, spine fusion, fracture repair andosteoporosis.

BACKGROUND OF THE INVENTION

Osteoblasts are thought to differentiate from pluripotent mesenchymalstem cells. The maturation of an osteoblast results in the secretion ofan extracellular matrix which can mineralize and form bone. Theregulation of this complex process is not well understood but is thoughtto involve a group of signaling glycoproteins known as bonemorphogenetic proteins (BMPs). These proteins have been shown to beinvolved with embryonic dorsal-ventral patterning, limb bud development,and fracture repair in adult animals. B. L. Hogan, Genes & Develop.,10:1580 (1996). This group of transforming growth factor-betasuperfamily secreted proteins has a spectrum of activities in a varietyof cell types at different stages of differentiation; differences inphysiological activity between these closely related molecules have notbeen clarified. D. M. Kingsley, Trends Genet., 10:16 (1994). To betterdiscern the unique physiological role of different BMP signalingproteins, we compared the potency of BMP-6 with that of BMP-2 and BMP4,for inducing rat calvarial osteoblast differentiation. Boden et al.,Endocrinology, 137:3401 (1996). We studied this process in first passage(secondary) cultures of fetal rat calvaria that require BMP orglucocorticoid for initiation of differentiation. In this model ofmembranous bone formation, glucocorticoid (GC) or a BMP will initiatedifferentiation to mineralized bone nodules capable of secretingosteocalcin, the osteoblast-specific protein. This secondary culturesystem is distinct from primary rat osteoblast cultures which undergospontaneous differentiation. In this secondary system, glucocorticoidresulted in a ten-fold induction of BMP-6 mRNA and protein expressionwhich was responsible for the enhancement of osteoblast differentiation.Boden et al., Endocrinology, 138:2920 (1997).

In addition to extracellular signals, such as the BMPs, intracellularsignals or regulatory molecules may also play a role in the cascade ofevents leading to formation of new bone. One broad class ofintracellular regulatory molecules are the LIM proteins, which are sonamed because they possess a characteristic structural motif known asthe LIM domain. The LIM domain is a cysteine-rich structural motifcomposed of two special zinc fingers that are joined by a 2-amino acidspacer. Some proteins have only LIM domains, while others contain avariety of additional functional domains. LIM proteins form a diversegroup, which includes transcription factors and cytoskeletal proteins.The primary role of LIM domains appears to be in mediatingprotein-protein interactions, through the formation of dimers withidentical or different LIM domains, or by binding distinct proteins.

In LIM homeodomain proteins, that is, proteins having both LIM domainsand a homeodomain sequence, the LIM domains function as negativeregulatory elements. LIM homeodomain proteins are involved in thecontrol of cell lineage determination and the regulation ofdifferentiation, although LIM-only proteins may have similar roles.LIM-only proteins are also implicated in the control of cellproliferation since several genes encoding such proteins are associatedwith oncogenic chromosome translocations.

Humans and other mammalian species are prone to diseases or injuriesthat require the processes of bone repair and/or regeneration. Forexample, treatment of fractures would be improved by new treatmentregimens that could stimulate the natural bone repair mechanisms,thereby reducing the time required for the fractured bone to heal. Inanother example, individuals afflicted with systemic bone disorders,such as osteoporosis, would benefit from treatment regimens that wouldresult in systemic formation of new bone. Such treatment regimens wouldreduce the incidence of fractures arising from the loss of bone massthat is a characteristic of this disease.

For at least these reasons, extracellular factors, such as the BMPs,have been investigated for the purpose of using them to stimulateformation of new bone in vivo. Despite the early successes achieved withBMPs and other extracellular signaling molecules, their use entails anumber of disadvantages. For example, relatively large doses of purifiedBMPs are required to enhance the production of new bone, therebyincreasing the expense of such treatment methods. Furthermore,extracellular proteins are susceptible to degradation following theirintroduction into a host animal. In addition, because they are typicallyimmunogenic, the possibility of stimulating an immune response to theadministered proteins is ever present.

In a pending U.S. application Ser. No. 11/385,612, filed by theassignee, the entire disclosures of all provisional and non-provisionalapplications are incorporated by reference herein, combinatorialtherapeutic strategies have been described including small molecules andpeptide mimics of LIM mineralization proteins. Eventhough the precisemechanism of LMP-1 is under investigation, it is generally thought thatexogenous BMPs induce bone formation by activating Smad1/5. Smad1/5 istargeted for degradation by Smurf1. The LMP-1 protein competes withSmad1/5 for Smurf1 binding thus increasing cellular responsiveness toexogenous BMPs.

Due to such concerns, it would be desirable to have available treatmentregimens that use an intracellular signaling molecule to induce new boneformation. Advances in the field of gene therapy now make it possible tointroduce into osteogenic precursor cells, that is, cells involved inbone formation, nucleotide fragments encoding intracellular signals thatform part of the bone formation process. Gene therapy for bone formationoffers a number of potential advantages: (1) lower production costs; (2)greater efficacy, compared to extracellular treatment regiments, due tothe ability to achieve prolonged expression of the intracellular signal;(3) it would by-pass the possibility that treatment with extracellularsignals might be hampered due to the presence of limiting numbers ofreceptors for those signals; (4) it permits the delivery of transfectedpotential osteoprogenitor cells directly to the site where localizedbone formation is required; and (5) it would permit systemic boneformation, thereby providing a treatment regimen for osteoporosis andother metabolic bone diseases.

SUMMARY OF THE INVENTION

The present invention seeks to overcome the drawbacks in the prior artby providing novel compositions and methods for inducing bone formationusing an intracellular signaling molecule that participates early in thecascade of events that leads to bone formation. Applicants havediscovered 10-4/RLMP (SEQ ID NO: 1, SEQ ID NO: 2), a novel LIM gene,homologous to a sequence originally isolated from stimulated ratcalvarial osteoblast cultures. The gene has been cloned, sequenced andassayed for its ability to enhance the efficacy of bone mineralizationin vitro. The protein RLMP affects mineralization of bone matrix as wellas differentiation of cells into the osteoblast lineage. Unlike otherknown cytokines, for example, BMPs, RLMP is not a secreted protein, butis instead an intracellular signaling molecule. This feature has theadvantage of providing intracellular signaling amplification as well aseasier assessment of transfected cells. It is also suitable for moreefficient and specific in vivo applications. Suitable clinicalapplications include enhancement of bone repair in fractures, bonedefects, bone grafting, and normal homeostasis in patients presentingwith osteoporosis.

Applicants have also cloned, sequenced and deduced the amino acidsequence of a corresponding human protein, named human LMP-1. The humanprotein demonstrates enhanced efficacy of bone mineralization in vitroand in vivo. LMP-1 contains an N-terminal PDZ domain and threeC-terminal LIM domains. Applicants have characterized a truncated(short) version of LMP-1, termed HLMP-1s, containing the N-terminal 223amino acids of the full length hLMP-1, while maintaining osteoinductiveactivity.

This short version resulted from a point mutation in one source of acDNA clone, providing a stop codon which truncated the protein. Theshort version (LMP-1s) is fully functional when expressed in cellculture and in vivo. In the invention instantly described, inventorshave assessed whether a truncated form of human LMP-1 [hLMP-1(t)],lacking the three C-terminal LIM domains, triggers differentiation ofpleuripotent myoblastic cells to the osteoblast lineage. One of theobjectives of the present invention is to show that at least one of theways in which osteoinductive proteins such as LMP-1 promote osteoblastdifferentiation involves up-regulation of multiple BMPs. Accordingly, auseful culture system is developed to evaluate the mechanism of actionof osteoinductive proteins such as LMP-1. A more preferred aspect ofthis invention is advancing methods of assessing the mechanisms ofaction of hLMP-1 in vitro.

Most preferably, the present invention provides a skeletal muscle cellculture model to assess the activity of LMP-1 and BMP signalingpathways. The present invention also offers those of ordinary skill inthe art the capability of conducting gene expression studies in myocytesand further screening for suitable factors or agents that can modulatethe growth factor signaling pathways.

Additional features and advantages of the invention will be set forth inthe description which follows, and in part will be apparent from thedescription, or may be learned by practice of the invention. Theobjectives and other advantages of the invention will be realized andattained by the methods and compositions of matter particularly pointedout in the written description and claims hereof.

In one broad aspect, the invention relates to an isolated nucleic acidmolecule comprising a nucleic acid sequence encoding any LIMmineralization protein, wherein the nucleic acid molecule hybridizesunder standard conditions to a nucleic acid molecule complementary tothe full length of SEQ. ID NO: 25, and wherein the molecule hybridizesunder highly stringent conditions to a nucleic acid moleculecomplementary to the full length of SEQ. ID NO: 26. In a specificaspect, the isolated nucleic acid molecule encodes HLMP-1, HLMP-1s orRLMP. In addition, the invention is directed to vectors comprising thesenucleic acid molecules, as well as host cells comprising the vectors. Inanother specific aspect, the invention relates to the proteinsthemselves.

In another aspect, the invention provides a method of switching adifferentiation of a cell from a non-osteogenic lineage into anosteogenic lineage comprising introducing into said cell an agentcapable of disrupting binding between a Smurf1 protein and a Smad1/5protein. In one embodiment, the step of introducing the agent comprisesoverexpressing a LMP protein or a fragment thereof in said cell.

In yet another aspect, the invention provides a method of treating abone void defect comprising: obtaining at least one cell of anon-osteogenic lineage; introducing into the at least one cell an agentcapable of disrupting binding between a Smurf1 protein and a Smad1/5protein; culturing the at least one cell for a time sufficient to directthe at least one cell to osteogenic lineage; introducing the at leastone cell of osteogenic lineage into a patient having the bone voiddefect. In one embodiment, the agent comprises a nucleic acid sequenceencoding an amino acid sequence which is at least 70% identical to theLMP protein or a fragment thereof. In different embodiments of theinvention, the amino acid sequence may be at least 75% identical, or atleast 80% identical, or at least 85% identical, or at least 90%identical, or at least 95% identical, or at least 995% identical, or100% identical to the LMP protein or the fragment thereof.

In another broad aspect, the invention relates to antibody that isspecific for LIM mineralization protein, including HLMP-1, HLMP-1s andRLMP. In one specific aspect, the antibody is a polyclonal antibody. Inanother specific aspect, the antibody is a monoclonal antibody.

In yet another aspect, the invention relates to method of inducing boneformation wherein osteogenic precursor cells are transfected with anisolated nucleic acid molecule comprising a nucleotide sequence encodingLIM mineralization protein. In one specific aspect, the isolated nucleicacid molecule is in a vector, which may be a plasmid or a virus, such asadenovirus or retrovirus. The transfection may occur ex vivo or in vivoby direct injection of the isolated nucleic acid molecule. Thetransfected isolated nucleic acid molecule may encode HLMP-1, HLMP-1s orRLMP.

In a further aspect, the invention relates to methods of fusing a spineby transfecting osteogenic precursor cells with an isolated nucleic acidmolecule having a nucleotide sequence encoding an amino acid sequence atleast 70% identical to the LIM mineralization protein, mixing thetransfected osteogenic precursor cells with a matrix and contacting thematrix with the spine.

In yet another aspect, the invention relates to methods for inducingsystemic bone formation by stable transfection of host cells with thevectors of the invention.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.

ABBREVIATIONS AND DEFINITIONS

-   -   BMP Bone Morphogenetic Protein    -   HLMP-1, Human LMP-1, also designated as Human LIM Protein or        HLMP or hLMP    -   HLMP-1s, hLMP-1(t), Human LMP-1 Short (truncated) protein    -   HLMPU, Human LIM Protein Unique Region    -   LMP, LIM mineralization protein    -   MEM, Minimal essential medium    -   Trm, Triamcinolone    -   β-GlyP, Beta-glycerolphosphate    -   RACE, Rapid Amplification of cDNA Ends    -   RLMP, Rat LIM mineralization protein, also designated as RLMP-1    -   RLMPU, Rat LIM Protein Unique Region    -   RNAsin, RNase inhibitor    -   ROB, Rat Osteoblast    -   10-4, Clone containing cDNA sequence for RLMP (SEQ ID NO: 2)    -   UTR, Untranslated Region    -   Primer Sequences for Ad5-GFP-LMP, mouse Alkaline Phosphatase,        Osteocalcin, BMP-2 and BMP-7.

Primer Sequence Endo-LMP-1 (forward) AGA GGG GCA CAT TGA AGT CCT TEndo-LMP-1 (reverse) CGA TCT GGC CAT ACA CTT GAG T Alkaline Phosphatase(forward) TCA GGG CAA TGA GGT CAC ATC Alkaline Phosphatase (reverse) CACAAT GCC CAC GGA CTT C Osteocalein (forward) CGG CCC TGA GTC TGA CAA AGOsteocalcin (reverse) CTC GTC ACA AGC AGG GTC AA BMP-2 (forward) CCG CTCCAC AAA CGA GAA A BMP-2 (reverse) CCA CAT CAC TGA AGT CCA CAT ACA BMP-7(forward) TGG CAC GTG ACG GAC AAG BMP-7 (reverse) GGA CAC TTT CCT GGCAGA CAT T

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel mammalian LIM proteins, hereindesignated LIM mineralization proteins, or LMP. The invention relatesmore particularly to human LMP, known as HLMP or HLMP-1.

LIM mineralization protein-1 (LMP-1), a novel intracellularosteoinductive protein, was cloned and sequenced in 1998. Boden et al.Endocrinology 139(12):5125-34 (1998), the teaching of which isincorporated herewith in its entirety. LMP-1 contains an N-terminal PDZdomain and three C-terminal LIM domains/motifs. David et al. 14:156-62(1998). The applicants have discovered that these proteins enhance bonemineralization in mammalian cells grown in vitro. When produced inmammals, LMP also induces bone formation in vivo.

Studies in osteoblast cultures have shown that LMP-1 does not requireLIM domains to induce bone formation as hLMP-1(t), a truncated humanLMP-1 containing the N-terminal 223 amino acids of the full lengthhLMP-1, (SEQ. ID. NO. 47) but not the LIM domains, was alsoosteoinductive in the above systems. Liu Y S et al. J Bone Min. Res.17:406-14 (2002), the teachings of which is incorporated herewith by itsentirety.

In the present invention, the inventors have determined whether the LIMdomains are required for LMP-1 osteoinductive activity in less committedpleuripotent cells and non-osteogenic lineages. The term “non-osteogeniclineage” throughout this application is used to characterize a cellwhich ordinarily does not develop into a cell exhibiting at least onecharacteristics of bone cells. Cells of non-osteogenic lineage include,for example, stem cells of limited pluripotency (e.g., tissue-specificstem cells, or satellite cells), such as, for example, myoblasts.

It is currently thought that exogenous BMPs induce bone formation byactivating Smad1/5. In this disclosure, the term “Smad1/5” refers toboth Smad1 and Smad5 proteins. Smad1/5 is targeted for degradation bySmurf1. LMP-1 protein competes with Smad1/5 for Smurf1 binding thusincreasing cellular responsiveness to exogenous BMPs. Sandagala et al.,Lim Mineralization Protein-1 Potentiates Bone Morphogenic ProteinResponsiveness via a Novel Interaction with Smurf1 resulting inDecreased Ubiquination of Smads, J. Biol. Chem. 281(25): 17212-17219(2006), incorporated herein by reference in its entirety. Withoutcommitting themselves to any particular theory, the applicants havediscovered a method of switching a differentiation pathway of a cellfrom a non-osteogenic lineage to an osteogenic lineage by introducing tothe cell an agent capable of disrupting binding between a Smurf1 proteinand a Smad1/5 protein. In one embodiment, the introducing of the agentcomprises overexpressing the LMP-1 protein or a fragment thereof such asa fragment which is necessary for binding the WW2 domain of the Smurf1protein and/or sufficient for osteogenic properties of the LMP-1protein. In some embodiments, the suitable fragments are included withinlarger amino acid sequences, which, may, optionally, be artificial.Suitable examples of such fragments and sequences include, withoutlimitations, SEQ. ID. NO. 48, SEQ. ID. NO. 49, SEQ. ID. NO. 50, and SEQ.ID. NO. 51.

In yet another embodiment, the nucleic acid sequence encodes an aminoacid sequence which is at least 70% identical to the LMP protein or thefragment thereof. A person of the ordinary skill will appreciate thatthe nucleic acid sequence encoding an amino acid sequence which may alsobe at least 75% identical, or at least 80% identical, or at least 85%identical, or at least 90% identical, or at least 95% identical, or atleast 99% identical, or 100% identical to the LMP protein or thefragment thereof.

In at least one aspect of this invention, inventors have developed auseful in vitro model system for examining the specificity andeffectiveness of the regulatory factors on the signaling pathways ofintracellular growth factors such as BMP-2 or TGF-β. In at least onepreferred embodiment of this invention, inventors show a novel use ofC₂C₁₂ cells in studying the LMP-1 induction of osteoblast lineage andits potential effects on the BMP-2 intracellular pathway.

Inventors have previously demonstrated that LMP-1 enhances bone noduleformation in committed fetal rat calvarial osteoblasts. However theseprimary cells must be taken from animals and expanded in culture inorder to perform each experiment. The biological responsiveness of theseprimary cells may change with the physiologic state of the cells whichvaries with different donors and different passages. In order to studythe mechanism of osteoinduction in induced osteoblast lineage, it isvery important to use a cell line on which osteoinductive proteins,preferably hLMP-1, has a consistent, predictable effect.

Inventors have chosen the C₂C₁₂ cells because they are pleuripotent.However, other cells of non-osteogenic lineage, such as myoblasts can bederived from an autologous, an allogenic, or a xenogenic source maybeemployed in the instant invention. Preferably, the cells ofnon-osteogenic lineage are derived from an autologous source, i.e., thepatient him- or herself. In one embodiment of the invention, a sample ofmuscle tissue may be obtained via a biopsy and the myoblasts areisolated.

The myoblasts can be isolated and substantially purified by isolationand purification schemes known in the art and described, for example, inWebster et al., Isolation of human myoblasts with thefluorescence-activated cell sorter, Exp Cell Res. 174(1):252-65 (1988),which is incorporated herein by reference in its entirety. That approachcapitalizes on the specific reaction of monoclonal antibody 5.1H11 witha human muscle cell surface antigen. Based on the results of thereactions, the myoblasts may be isolated using thefluorescence-activated cell sorter. A fraction of myoblasts to greaterthan 99% of the cell population can be achieved. An average of 10⁴viable myoblasts can be obtained per 0.1 g tissue, each with thepotential to undergo approximately 40 cell divisions.

In one embodiment of the instant invention, C₂C₁₂ cells were originallyisolated from injured adult mouse muscle and have proven an excellentmodel system for studying myogenic differentiation. C₂C₁₂ cellsproliferate in culture medium, but undergo terminal differentiation whengrown to confluence and deprived of growth factors. During this process,C₂C₁₂ cells exit the cell cycle, up-regulate muscle-specific genes, andfuse into multinucleated myotubes. Yaffe et al. Nature; 270:725-27(1997). C₂C₁₂ cells can differentiate into the osteoblast phenotype iftreated with cytokines, such as bone morphogenetic protein-2 (BMP-2).Ying et al., JBC; 278:39029-036 (2003).

In this embodiment of the instant invention, inventors study theinduction of the osteoblast lineage from pleuripotent cells when saidcells are treated with intracellular growth factor, preferably,osteoinductive proteins, and more preferably isoforms of LMP1, such astruncated human LMP-1. Inventors have further assessed the potentialeffects of said factors on the BMP signaling pathway.

According to this aspect of the invention, the inventors are the firstto show the advantages of these pleuripotent cells (i.e. mammalian C₂C₁₂myocytes) over primary cells, such as bone marrow cells and ratcalvarial osteoblasts. The most striking finding of this aspect of theinvention is the induction of pleuripotent myoblastic cells to becomeosteoblastic cells as measured by altered morphology and osteoblastspecific gene expression. This observation is the first demonstrationthat an LMP-1 isoform can drive any pleuripotent cell toward theosteoblast phenotype.

As a feature of this invention inventors compared the binding ofbiotinylated full length and truncated human LMP-1 to nuclear proteinsin a biotin-transfer assay to validate using the truncated hLMP-1 andthe effects thereof on C₂C₁₂ cultures. Inventors also identified thatboth full length LMP-1 and LMP-1(t) interact with Smurf1 suggesting thatthey induce osteoblast differentiation by similar mechanisms.

In another embodiment, the cells may be isolated by a combination ofmagnetic-activated cell sorting (MACS), complement-mediatedcytotoxicity, and a preplating technique, as described, for example, inPark et al., A Comparative Study of Magnetic-Activated Cell Sorting,Cytotoxicity and Preplating for the Purification of Human Myoblasts,Yonsei Med J., 47(2):179-183 (2006). The authors report that using thatcombination they obtained a cell culture comprising 92.8% of myoblasts.

Additional criteria for selecting the cells with desired characteristicsare known in the art and include, for example, high proliferativecapacity, as discussed in Jankowski et al., Establishing reliablecriteria for isolating myogenic cell fractions with stem cell propertiesand enhanced regenerative capacity, Blood Cell Mol Dis, 3291):24-33(2004). This and other criteria may advantageously be included into theprotocols described above.

In another embodiment of this invention, inventors transferred biotin toa protein with which either forms of LMP interacts. The newlybiotinylated LMP-1-interacting proteins were then separated by SDS-PAGEand the biotin label detected using neutravadin-HRP. Accordingly,Inventors found that there was no difference in the detected bands,suggesting that full length and truncated hLMP-1 have the same bindingpartners and are likely to exert similar physiologic effects A 85 kDaband was determined to be Smurf1; another two bands at 50 kDa and 55 kDawere determined to be meosin and caldesmon, respectively.

In another aspect of the instant invention, inventors were able todetermine the efficiency of bone growth factors transduction in C₂C₁₂cultures. In at least one preferred embodiment, the inventors determinedthe efficiency of Ad5-hLMP-1(t)-GFP transduction of C₂C₁₂ cultures andthus identified whether truncated human LMP-1 induces pleuripotent cellsto the osteoblast lineage.

To achieve such goal, the inventors constructed hLMP-1(t) cDNA whichcodes for a protein containing the N-terminal 223 amino acids of LMP-1,but not the LIM domains. Inventors then delivered the cDNA viaadenoviral delivery to improve transduction efficiency and assessinduction of gene expression in C₂C₁₂ cultures by hLMP-1(t)overexpression. Flow cytometry or other known methods to one of ordinaryskill in the art was employed to identify cells that were taken up thevector. Accordingly, it was found that the number of cells expressingGFP increased as increasing amounts of virus were added to the culturein the dose range between 10 and 500 pfu/cell.

In another embodiment of the present invention, the optimal dose ofvirus to achieve maximum expression of hLMP-1(t) was determinedAccordingly, the inventors performed an experiment in which variousdoses of Ad5-hLMP-1(t)-GFP were added and the level of LMP-1 mRNAmeasured four days later by real time RT-PCR.

Primers were designed that detect human overexpressed LMP-1 mRNA, butnot endogenous mouse LMP-1. This strategy allows one of ordinary skillin the art to demonstrate overexpression of human LMP-1 even at thelowest dose tested (10 pfu/cell). For the first time, one of ordinaryskill in the art can appreciate the ability to demonstrate suchoverexpression.

The real-time PCR analysis showed that transduced human LMP-1 mRNA wasabundantly expressed. The relative value of hLMP-1(t) gene expression inthe transfected cells was much higher than the control because no mouseLMP-1 mRNA was detected in the control C₂C₁₂ cells by this method.Inventors determined that LMP-1 expression increased as increasingamounts of Ad5-hLMP-1(t)-GFP were added to C₂C₁₂ cultures in the rangeof 10-250 pfu/cell. In subsequent experiments 100 pfu/cell was appliedbecause that is the lowest dose giving maximum expression of hLMP-1(t).

Western blot analysis has further confirmed a prominent signal oftruncated LMP-1 at about 30. According to this aspect of the invention,a western blot analysis was performed on the proteins extracted fromC₂C₁₂ cultures transduced with 100 pfu/cell Ad5-hLMP-1(t)-GFP usingaffinity purified hLMP-1 specific antibody. A band at approximately 50kDa was determined to most likely represent endogenous mouse LMP-1.

In another embodiment, the inventors have shown for the first time thatintracellular growth factor, preferably, osteoinductive proteins, andmore preferably isoforms of LMP1, such as truncated human LMP-1, areable to block C₂C₁₂ cell myotube formation and enhance its osteoblasticcharacteristics. More specifically, those of ordinary skill in the artcan now appreciate that C₂C₁₂ cells have the potential to differentiateinto myoblasts when cultured in appropriate medium.

Under these control conditions, many myotubes can be seen under aphase-contrast microscope. However C₂C₁₂ cultures transduced withAd5-GFP-LMP-1(t), have very few myotubes four days after transduction,indicating that hLMP-1(t) has an inhibitory effect on C₂C₁₂ myotubeformation.

To determine whether the transduced cultures had differentiated towardthe osteoblastic phenotype, alkaline phosphatase activity was measured.Overexpression of hLMP-1(t) for 4 days induced a 50% increase inalkaline phosphatase activity in C₂C₁₂ cultures compared with culturestreated with the empty vector control.

Increased expression of the alkaline phosphatase and osteocalcin genesare markers of osteoblastic differentiation. In C₂C₁₂ culturesoverexpressing hLMP-1(t) for four days, alkaline phosphatase andosteocalcin gene expression was significantly increased 9 and 13-fold,respectively, as compared with the control C₂C₁₂ cultures treated withvector alone. In order to learn more regarding the possible mechanism ofhLMP-1(t) induction of osteoblastic differentiation in C₂C₁₂ cultures,BMP-2 and BMP-7 gene expression were also measured four days aftertransduction. As it is apparent to those of ordinary skill in the art,inventors of the present invention show that expression of both geneswas significantly increased following over-expression of hLMP-1(t). Alsosee Minamide, et al. J Bone & Joint Surg Am 85A:1030-39 (2003), theteachings of which is incorporated by its entirety herein with.

In hLMP-1(t) overexpressing cultures, BMP-2 expression was increased8-fold over the vector control, while BMP-7 expression increased 10-foldin those cultures. Taken together, the data proves to those of ordinaryskill in the art that overexpression of hLMP-1(t) inhibits the myoblastphenotype in differentiating C₂C₁₂ cultures while enhancing expressionof several markers of the osteoblastic phenotype.

Those of ordinary skill in the art would appreciate the striking findingof the present invention, wherein pleuripotent myoblastic cells havebeen induced to become osteoblastic cells as measured by alteredmorphology and osteoblast specific gene expression. This observation isthe first demonstration that osteoinductive proteins, preferably, anLMP-1 isoform, can drive any pleuripotent cell toward the osteoblastphenotype.

It has been shown that overexpressed full length or truncated rat orhuman LMP-1 can enhance the ability of committed preosteoblasts (primarycultures isolated from rat calvariae) to express osteoblastic markersand form bone nodules. see Boden et al., 139 (12):5125-34 (1998) and Liuet al., J Bone Min. Res. 17:406-14 (2002).

In at least one aspect of the present invention, one of ordinary skillin the art would appreciate understanding the finding that thatpleuripotent cells with a myoblastic phenotype are induced to osteoblastphenotype when treated with overexpressed osteoinductive proteins suchas truncated hLMP-1.

In the most preferred embodiment of the present invention, C₂C₁₂cultures transiently transduced with Ad5-hLMP1(t)-GFP weredifferentiated to the osteoblastic phenotype four days later asevidenced by up-regulation of osteocalcin and alkaline phosphatase geneexpression. In the control groups, C₂C₁₂ cells differentiated to themyoblast phenotype and, as expected, did not increase osteocalcin andalkaline phosphatase gene expression. Hence C₂C₁₂ cells are anappropriate model system for examining the mechanisms of LMP inductionof the osteoblast phenotype and further screening suitable candidateagents that can modulate said pathways. Accordingly, such an in vitrodifferentiation process offers a very useful model system for examiningthe specificity and effectiveness of regulatory factors on TGF-β orBMP-2 signaling.

The present invention also offers those of ordinary skill in the art thecapability of conducting gene expression studies and further screeningfor suitable agents that can modulate the growth factor signalingpathways.

In yet another embodiment of the present invention, ex vivo transfectionof bone marrow cells, osteogenic precursor cells or mesenchymal stemcells with nucleic acid that encodes LMP or HLMP, followed byreimplantation of the transfected cells in the donor, is envisioned tobe suitable for treating a variety of bone-related disorders orinjuries. For example, one can use this method to: augment long bonefracture repair; generate bone in segmental defects; provide a bonegraft substitute for fractures; facilitate tumor reconstruction or spinefusion; and provide a local treatment (by injection) for weak orosteoporotic bone, such as in osteoporosis of the hip, vertebrae, orwrist.

Transfection with LMP or HLMP-encoding nucleic acid is also useful inthe percutaneous injection of transfected marrow cells to accelerate therepair of fractured long bones; treatment of delayed union or non-unionsof long bone fractures or pseudoarthrosis of spine fusions; and forinducing new bone formation in avascular necrosis of the hip or knee. Inat least one embodiment of this invention, either before or afterintroducing the nucleic acid encoding the LMP-1 protein or a fragmentthereof, the isolated cells may be cultured on a substrate shapeableinto a shape of the bone void. Suitable substrates include, withoutlimitations, cages or meshes, which may optionally be coated withcompositions to induce cell attachment. A variety of these compositionsare known in the art and commercially available. A suitable example ofsuch composition includes Matrigel™, available from BD Biosciences, Inc.(San Jose, Calif.).

In addition to ex vivo-based methods of gene therapy, transfection of arecombinant DNA vector comprising a nucleic acid sequence that encodesLMP or HLMP can be accomplished in vivo. When a DNA fragment thatencodes LMP or HLMP is inserted into an appropriate viral vector, forexample, an adenovirus vector, the viral construct can be injecteddirectly into a body site were endochondral bone formation is desired.By using a direct, percutaneous injection to introduce the LMP or HLMPsequence stimulation of bone formation can be accomplished without theneed for surgical intervention either to obtain bone marrow cells (totransfect ex vivo) or to reimplant them into the patient at the sitewhere new bone is required. Alden et al., Neurosurgical Focus (1998),have demonstrated the utility of a direct injection method of genetherapy using a cDNA that encodes BMP-2, which was cloned into anadenovirus vector.

It is also possible to carry out in vivo gene therapy by directlyinjecting into an appropriate body site, a naked, that is,unencapsulated, recombinant plasmid comprising a nucleic acid sequencethat encodes HLMP. In this embodiment of the invention, transfectionoccurs when the naked plasmid DNA is taken up, or internalized, by theappropriate target cells, which have been described. As in the case ofin vivo gene therapy using a viral construct, direct injection of nakedplasmid DNA offers the advantage that little or no surgical interventionis required. Direct gene therapy, using naked plasmid DNA that encodesthe endothelial cell mitogen VEGF (vascular endothelial growth factor),has been successfully demonstrated in human patients. Baumgartner etal., Circulation, 97(12):1114-23 (1998).

By using an adenovirus vector to deliver LMP into osteogenic cells,transient expression of LMP is achieved. This occurs because adenovirusdoes not incorporate into the genome of target cells that aretransfected. Transient expression of LMP, that is, expression thatoccurs during the lifetime of the transfected target cells, issufficient to achieve the objects of the invention. Stable expression ofLMP, however, can occur when a vector that incorporates into the genomeof the target cell is used as a delivery vehicle. Retrovirus-basedvectors, for example, are suitable for this purpose.

Stable expression of LMP is particularly useful for treating varioussystemic bone-related disorders, such as osteoporosis and osteogenesisimperfecta. For this embodiment of the invention, in addition to using avector that integrates into the genome of the target cell to deliver anLMP-encoding nucleotide sequence into target cells, LMP expression isplaced under the control of a regulatable promoter. For example, apromoter that is turned on by exposure to an exogenous inducing agent,such as tetracycline, is suitable. Using this approach, one canstimulate formation of new bone on a systemic basis by administering aneffective amount of the exogenous inducing agent. Once a sufficientquantity of bone mass is achieved, administration of the exogenousinducing agent is discontinued. This process may be repeated as neededto replace bone mass lost, for example, as a consequence ofosteoporosis.

Antibodies specific for HLMP are particularly suitable for use inmethods for assaying the osteoinductive, that is, bone-forming,potential of patient cells. In this way one can identify patients atrisk for slow or poor healing of bone repair. Also, HLMP-specificantibodies are suitable for use in marker assays to identify riskfactors in bone degenerative diseases, such as, for example,osteoporosis.

Following well known and conventional methods, the genes of the presentinvention are prepared by ligation of nucleic acid segments that encodeLMP to other nucleic acid sequences, such as cloning and/or expressionvectors. Methods needed to construct and analyze these recombinantvectors, for example, restriction endonuclease digests, cloningprotocols, mutagenesis, organic synthesis of oligonucleotides and DNAsequencing, have been described. For DNA sequencing DNA, thedieoxyterminator method is the preferred.

Many treatises on recombinant DNA methods have been published, includingSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, 2nd edition (1988), Davis et al., Basic Methods inMolecular Biology, Elsevier (1986), and Ausubel et al., CurrentProtocols in Molecular Biology, Wiley Interscience (1988). Thesereference manuals are specifically incorporated by reference herein.

Primer-directed amplification of DNA or cDNA is a common step in theexpression of the genes of this invention. It is typically performed bythe polymerase chain reaction (PCR). PCR is described in U.S. Pat. No.4,800,159 to Mullis et al. and other published sources. The basicprinciple of PCR is the exponential replication of a DNA sequence bysuccessive cycles of primer extension. The extension products of oneprimer, when hybridized to another primer, becomes a template for thesynthesis of another nucleic acid molecule. The primer-templatecomplexes act as substrate for DNA polymerase, which in performing itsreplication function, extends the primers. The conventional enzyme forPCR applications is the thermostable DNA polymerase isolated fromThermus aquaticus, or Taq DNA polymerase.

Numerous variations of the basic PCR method exist, and a particularprocedure of choice in any given step needed to construct therecombinant vectors of this invention is readily performed by a skilledartisan. For example, to measure cellular expression of 10-4/RLMP, RNAis extracted and reverse transcribed under standard and well knownprocedures. The resulting cDNA is then analyzed for the appropriate mRNAsequence by PCR.

The gene encoding the LIM mineralization protein is expressed in anexpression vector in a recombinant expression system. Of course, theconstructed sequence need not be the same as the original, or itscomplementary sequence, but instead may be any sequence determined bythe degeneracy of the DNA code that nonetheless expresses an LMP havingbone forming activity. Conservative amino acid substitutions, or othermodifications, such as the occurrence of an amino-terminal methionineresidue, may also be employed.

A ribosome binding site active in the host expression system of choiceis ligated to the 5′ end of the chimeric LMP coding sequence, forming asynthetic gene. The synthetic gene can be inserted into any one of alarge variety of vectors for expression by ligating to an appropriatelylinearized plasmid. A regulatable promoter, for example, the E. coli lacpromoter, is also suitable for the expression of the chimeric codingsequences. Other suitable regulatable promoters include trp, tac, recA,T7 and lambda promoters.

DNA encoding LMP is transfected into recipient cells by one of severalstandard published procedures, for example, calcium phosphateprecipitation, DEAE-Dextran, electroporation or protoplast fusion, toform stable transformants. Calcium phosphate precipitation is preferred,particularly when performed as follows.

DNAs are coprecipitated with calcium phosphate according to the methodof Graham and Van Der, Virology, 52:456 (1973), before transfer intocells. An aliquot of 40-50 μg of DNA, with salmon sperm or calf thymusDNA as a carrier, is used for 0.5×10⁶ cells plated on a 100 mm dish. TheDNA is mixed with 0.5 ml of 2×Hepes solution (280 mM NaCl, 50 mM Hepesand 1.5 mM Na₂HPO₄, pH 7.0), to which an equal volume of 2.×CaCl₂ (250mM CaCl₂ and 10 mM Hepes, pH 7.0) is added. A white granularprecipitate, appearing after 30-40 minutes, is evenly distributeddropwise on the cells, which are allowed to incubate for 4-16 hours at37° C. The medium is removed and the cells shocked with 15% glycerol inPBS for 3 minutes. After removing the glycerol, the cells are fed withDulbecco's Minimal Essential Medium (DMEM) containing 10% fetal bovineserum.

DNA can also be transfected using: the DEAS-Dextran methods of Kimura etal., Virology, 49:394 (1972) and Sompayrac et al., Proc. Natl. Acad.Sci. USA, 78:7575 (1981); the electroporation method of Potter, Proc.Natl. Acad. Sci. USA, 81:7161 (1984); and the protoplast fusion methodof Sandri-Goddin et al., Molec. Cell. Biol., 1:743 (1981).

Phosphoramidite chemistry in solid phase is the preferred method for theorganic synthesis of oligodeoxynucleotides and polydeoxynucleotides. Inaddition, many other organic synthesis methods are available. Thosemethods are readily adapted by those skilled in the art to theparticular sequences of the invention.

The present invention also includes nucleic acid molecules thathybridize under standard conditions to any of the nucleic acid sequencesencoding the LIM mineralization proteins of the invention. “Standardhybridization conditions” will vary with the size of the probe, thebackground and the concentration of the nucleic acid reagents, as wellas the type of hybridization, for example, in situ, Southern blot, orhybridization of DNA-RNA hybrids (Northern blot). The determination of“standard hybridization conditions” is within the level of skill in theart. For example, see U.S. Pat. No. 5,580,775 to Fremeau et al., hereinincorporated by reference for this purpose. See also, Southern, E. M.,J. Mol. Biol., 98:503 (1975), Alwine et al., Meth. Enzymol., 68:220(1979), and Sambrook et al., Molecular Cloning: A laboratory Manual, 2ndedition, pp. 7.19-7.50, Cold Spring Harbor Press (1989).

One preferred set of standard hybridization conditions involves a blotthat is prehybridized at 42° C. for 2 hours in 50% formamide, 5×SSPE(150 nM NaCl, 10 mM Na H₂PO₄ [pH 7.4], 1 mM EDTA [pH 8.0]), 5×Denhardt'ssolution (20 mg Ficoll, 20 mg polyvinylpyrrolidone and 20 mg BSA per 100ml water), 10% dextran sulphate, 1% SDS and 100 μg/ml salmon sperm DNA.A ³²P-labeled cDNA probe is added, and hybridization is continued for 14hours. Afterward, the blot is washed twice with 2×SSPE, 0.1% SDS for 20minutes at 22° C., followed by a 1 hour wash at 65° C. in 0.1×SSPE, 0.1%SDS. The blot is then dried and exposed to x-ray film for 5 days in thepresence of an intensifying screen.

Under “highly stringent conditions,” a probe will hybridize to itstarget sequence if those two sequences are substantially identical. Asin the case of standard hybridization conditions, one of skill in theart can, given the level of skill in the art and the nature of theparticular experiment, determine the conditions under which onlysubstantially identical sequences will hybridize.

Another aspect of the invention includes the proteins encoded by thenucleic acid sequences. In still another embodiment, the inventionrelates to the identification of such proteins based on anti-LMPantibodies. In this embodiment, protein samples are prepared for Westernblot analysis by lysing cells and separating the proteins by SDS-PAGE.The proteins are transferred to nitrocellulose by electroblotting asdescribed by Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons (1987). After blocking the filter with instantnonfat dry milk (5 gm in 100 ml PBS), anti-LMP antibody is added to thefilter and incubated for 1 hour at room temperature. The filter iswashed thoroughly with phosphate buffered saline (PBS) and incubatedwith horseradish peroxidase (HRPO)-Anti-rabbit antibody conjugate for 1hour at room temperature. The filter is again washed thoroughly-with PBSand the antigen bands are identified by adding diaminobenzidine (DAB).

Monospecific antibodies are the reagent of choice in the presentinvention, and are specifically used to analyze patient cells forspecific characteristics associated with the expression of LMP.“Monospecific antibody” as used herein is defined as a single antibodyspecies or multiple antibody species with homogenous bindingcharacteristics for LMP as they are purified using LMP-affinity column.“Homogeneous binding” as used herein refers to the ability of theantibody species to bind to a specific antigen or epitope, such as thoseassociated with LMP, as described above. Monospecific antibodies to LMPare purified from mammalian antisera containing antibodies reactiveagainst LMP or are prepared as monoclonal antibodies reactive with LMPusing the technique of Kohler and Milstein, Nature, 256:495-97 (1975).The LMP specific antibodies are raised by immunizing animals such as,for example, mice, rats, guinea pigs, rabbits, goats or horses, with anappropriate concentration of LMP either with or without an immuneadjuvant.

In this process, preimmune serum is collected prior to the firstimmunization. Each animal receives between about 0.1 mg and about 1.0mg, preferably about 1 mg, of LMP associated with an acceptable immuneadjuvant, if desired. Such acceptable adjuvants include, but are notlimited to, Freund's complete, Freund's incomplete, alum-precipitate,water in oil emulsion containing Corynebacterium parvum and tRNAadjuvants. The initial immunization consists of LMP in, preferably,Freund's complete adjuvant injected at multiple sites eithersubcutaneously (SC), intraperitoneally (IP) or both. Each animal is bledat regular intervals, preferably weekly, to determine antibody titer.The animals may or may not receive booster injections following theinitial immunization. Those animals receiving booster injections aregenerally given an equal amount of the antigen in Freund's incompleteadjuvant by the same route. Booster injections are given at about threeweek intervals until maximal titers are obtained. At about 7 days aftereach booster immunization or about weekly after a single immunization,the animals are bled, the serum collected, and aliquots are stored atabout −20° C.

Monoclonal antibodies (mAb) reactive with LMP are prepared by immunizinginbred mice, preferably Balb/c mice, with LMP. The mice are immunized bythe IP or SC route with about 0.1 mg to about 1 mg, preferably about 1mg, of LMP in about 0.5 ml buffer or saline incorporated in an equalvolume of an acceptable adjuvant, as discussed above. Freund's completeadjuvant is preferred. The mice receive an initial immunization on day 0and are rested for about 3-30 weeks. Immunized mice are given one ormore booster immunizations of about 0.1 to about 1 mg, preferably about1 mg, of LMP in a buffer solution such as phosphate buffered saline bythe intravenous (IV) route. Lymphocytes from antibody-positive mice,preferably splenic lymphocytes, are obtained by removing the spleensfrom immunized mice by standard procedures known in the art. Hybridomacells are produced by mixing the splenic lymphocytes with an appropriatefusion partner, preferably myeloma cells, under conditions which willallow the formation of stable hybridomas. Fusion partners may include,but are not limited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 andSp 2/0, with Sp 2/0 being preferred. The antibody producing cells andmyeloma cells are fused in polyethylene glycol, about 1000 mol. wt., atconcentrations from about 30% to about 50%. Fused hybridoma cells areselected by growth in hypoxanthine, thymidine and aminopterin insupplemented Dulbecco's Modified Eagles Medium (DMEM) by proceduresknown in the art. Supernatant fluids are collected from growth positivewells on about days 14, 18, and 21, and are screened for antibodyproduction by an immunoassay such as solid phase immunoradioassay(SPIRA) using LMP as the antigen. The culture fluids are also tested inthe Ouchterlony precipitation assay to determine the isotype of the mAb.Hybridoma cells from antibody positive wells are cloned by a techniquesuch as the soft agar technique of MacPherson, “Soft Agar Techniques”,in Tissue Culture Methods and Applications, Kruse and Paterson (eds.),Academic Press (1973). See, also, Harlow et al., Antibodies: ALaboratory Manual, Cold Spring Laboratory (1988).

Monoclonal antibodies may also be produced in vivo by injection ofpristane-primed Balb/c mice, approximately 0.5 ml per mouse, with about2×10⁶ to about 6×10⁶ hybridoma cells about 4 days after priming. Ascitesfluid is collected at approximately 8-12 days a52fter cell transfer andthe monoclonal antibodies are purified by techniques known in the art.

In vitro production in anti-LMP mAb is carried out by growing thehydridoma cell line in DMEM containing about 2% fetal calf serum toobtain sufficient quantities of the specific mAb. The mAb are purifiedby techniques known in the art.

Antibody titers of ascites or hybridoma culture fluids are determined byvarious serological or immunological assays, which include, but are notlimited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (ELISA) technique and radioimmunoassay (RIA)techniques. Similar assays are used to detect the presence of the LMP inbody fluids or tissue and cell extracts.

It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for polypeptide fragments of LMP, fulllength nascent LMP polypeptide, or variants or alleles thereof.

On Jul. 22, 1997, a sample of 10-4/RLMP in a vector designatedpCMV2/RLMP (which is vector pRc/CMV2 with insert 10-4 clone/RLMP) wasdeposited with the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md. 20852. The culture accession number forthat deposit is 209153. On Mar. 19, 1998, a sample of the vector pHis-Awith insert HLPM-1s was deposited at the American Type CultureCollection. The culture accession number for that deposit is 209698.These deposits, made under the requirements of the Budapest Treaty, willbe maintained in the ATCC for at least 30 years and will be madeavailable to the public upon the grant of a patent disclosing them. Itshould be understood that the availability of a deposit does notconstitute a license to practice the subject invention in derogation ofpatent rights granted by government action.

In assessing the nucleic acids, proteins, or antibodies of theinvention, enzyme assays, protein purification, and other-conventionalbiochemical methods are employed. DNA and RNA are analyzed by Southernblotting and Northern blotting techniques, respectively. Typically, thesamples analyzed are size fractionated by gel electrophoresis. The DNAor RNA in the gels are then transferred to nitrocellulose or nylonmembranes. The blots, which are replicas of sample patterns in the gels,were then hybridized with probes. Typically, the probes areradiolabelled, preferably with ³²P, although one could label the probeswith other signal-generating molecules known to those in the art.Specific bands of interest can then be visualized by detection systems,such as autoradiography. For purposes of illustrating preferredembodiments of the present invention, the following, none limitingexamples are included. These results demonstrate the feasibility ofinducing or enhancing the formation of bone using the LIM mineralizationproteins of the invention, and the isolated nucleic acid moleculesencoding those proteins.

EXAMPLES Example 1

Materials—C₂C₁₂ cells and Dulbecco's Modified Eagle's Medium (DMEM) werepurchased from ATCC (Manassas, Va.). Fetal bovine serum was from AtlantaBiologicals (Atlanta, Ga.). Lipofectamine, Opti-MEM, NuPAGE™ 4-12%Bis-Tris Gel and polyvinylidene difluoride membrane were purchased fromInvitrogen (Carlsbad, Calif.). The Alkaline Phosphatase (ALP) assay kitwas from Sigma-Aldrich (St. Louis, Mo.). The Bio-rad protein assay kitwas from Bio-Rad Laboratory (Hercules, Calif.). Horseradishperoxidase-labeled goat anti-rabbit IgG and western lightening reagent Aand B were purchased from Perkin Elmer Life Science (Boston, Mass.).RNeasy mini kit and DNAse 1 were from QIAGEN Inc. (Valencia, Calif.).Reverse transcription reagents and SYBR Green Real-Time PCR Kit werepurchased from Applied Biosystems (Foster City, Calif.).

Example 2

Cell culture—C₂C₁₂ cells (ATCC, Manassas, Va.) at passage 3 or 4 weresubcultured in T-75 cm² flasks (Corning, Inc., Corning, N.Y.) inDulbecco's Modified Eagle's Medium (DMEM; ATCC, Manassas, Va.)supplemented with 10% fetal bovine serum (Atlanta Biologicals, Atlanta,Ga.) at 37° C. in 5% CO₂ with humidification. When flasks were 80%confluent, cells were trypsinized and passed to 6-well plates at 200,000cells per well (2×10⁴ cells/cm²).

Example 3

Ad5-hLMP1(t)-GFP construction—The procedure for construction ofAd5-hLMP1(t) was previously described. To prepare Ad5-hLMP-1(t)-GFP weused a shuttle vector (QBiogene, Montreal, Quebec, Canada) thatcontained GFP cDNA downstream of an internal ribosomal entry site(IRES). Truncated human LMP-1 was cloned into the multiple cloning sitelocated upstream of the IRES. Recombination occurred in 293 cellsbetween the homologous regions of the linearized transfer vector and theadenovirus genome, resulting in the formation of the complete adenoviralrecombinant (Ad5-LMP-1(t)-GFP). The recombinant adenovirus was selected,further amplified, and purified by cesium chloride gradientcentrifugation and titered by plaque assay. The viral titer was 9×10⁹pfu/ml.

Example 4

Biotin transfer assay for detection of LMP-1/LMP-1(t) interactingproteins—Sulfo-SBED (Pierce, Rockford, Ill.), a trifunctionalcross-linking agent, contains three functional groups (aphotoactivatable aryl azide, a sulfonated N-hydroxy succinimide activeester with a cleavable disulfide group and a biotin moiety) and iswidely used to identify interacting proteins LMP-1 or LMP-1(t) waslabeled using this reagent, incubated as bait with nuclear proteins andcross-linked to interacting proteins by UV (365 nm). Proteins thatphysically interact with LMP-1 or LMP-1(t) retain the biotin group whensuspended in SDS-PAGE reducing buffer. Biotin-containing target proteinswere separated using neutravidin beads, detected by Western blottingwith neutravidin-HRP and the signal was developed with chemiluminescentsubstrate. Corresponding protein bands were in-gel digested withtrypsin. Tryptic peptides were recovered, concentrated and theirtryptic-peptide mass profile was analyzed by MALDI-TOF at the EmoryUniversity Microchemical Facility.

Example 5

Transient Ad5-hLMP1(t)-GFP and Ad5-GFP transduction and morphologicobservations—One day after C₂C₁₂ cells were sub-cultured in 6-wellplates (200,000 cells/well), cells were treated with Lipofectamine in500 ul Opti-MEM per well (Invitrogen, Carlsbad, Calif.) for 4 hours topromote adenovirus transduction efficiency. They were then transducedwith Ad5-hLMP1(t)-GFP or Ad5-GFP (as control) in 300 ul Opti-MEM(Invitrogen, Carlsbad, Calif.) for 30 minutes. After transductionOpti-MEM was added to 2 ml/well and the cells were incubated at 37° C.After two days the medium was changed to DMEM supplemented with 10%fetal bovine serum and the cells were allowed to differentiate foranother 2 days. Dose/response experiments over the range of 10-500pfu/cell were performed and the cells or total RNA was harvested todetermine either the transduction efficiency by flow cytometry or thedose of Ad5-hLMP-1(t)-GFP resulting in maximal expression of LMP-1 byreal time RT-PCR. Based on the outcome of those experiments, 100pfu/cell was applied to cultures in subsequent studies. In each suchexperiment, the cells from one well were harvested on day four for mRNAanalysis; cells from another two parallel wells were harvested foralkaline phosphatase activity and protein analysis. Before the cellswere harvested, the cell morphology was observed under a phase-contrastmicroscope and photographed.

Example 6

Alkaline phosphatase activity and protein assay—After being washed twicewith ice-cold phosphate-buffered saline, the cells were lysed bysonication in lysis buffer (10 mM Tris pH 8.0, 1 mM MgCl₂, 0.5% TritonX-100). The cell lysates were centrifuged and the supernatants wereisolated for analysis of alkaline phosphatase activity and proteinlevels. Alkaline phosphatase activity in cell lysates was measured usinga Sigma ALP assay kit (Sigma, St. Louis, Mo.). Protein content wasmeasured using the Bio-Rad protein assay kit (Bio-Rad Laboratory,Hercules, Calif.) using a BSA standard curve. The alkaline phosphataseactivity and protein content were measured in triplicate and thealkaline phosphatase activity was normalized to protein content.

Example 7

Western blot analysis of truncated human LMP-1 overexpression—Todemonstrate the presence of truncated human LMP-1 protein in transducedC₂C₁₂ cells, the cell lysates that were prepared for alkalinephosphatase measurement (20 ug per sample) were separated on NuPAGE™4-12% Bis-Tris Gels (Invitrogen, Carlsbad, Calif.) and transferred ontoa polyvinylidene difluoride membrane (Invitrogen, Carlsbad, Calif.). Themembrane was first blocked with 5% milk for 1 hour and then incubatedwith affinity-purified rabbit anti-LMP1 antibody (1:2500 dilution) andsecondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG1:5,000 dilution, Perkin Elmer Life Science, Boston, Mass.) for 1 hour,respectively, at room temperature. The signal was then developed usingchemiluminescent substrates (western lightening reagent A and B mixture;Perkin Elmer Life Science, Boston, Mass.).

Example 8

Quantitative real time RT-PCR measurements of gene expression—RNA andcDNA Preparation: Total RNA from each sample was extracted using theRNeasy mini kit, following the RNeasy mini protocol for isolation oftotal RNA from animal cells (QIAGEN Inc., Valencia, Calif.). Theisolated RNA was treated with DNAse 1 (QIAGEN Inc., Valencia, Calif.) toremove DNA contamination from the samples. The concentration of theisolated RNA was spectrophotometrially determined (DU-500; Beckman,Fullerton, Calif.) at a 260-nm wavelength and protein contamination wasdetermined at a 280 wavelength. The ratio of 260/280 was between1.6˜1.8. Reverse transcription was carried out in 100 μL volume with 1μg of total RNA, 10 μL 10×RT buffer, 5.5 mM MgCl₂, 2 mM dNTP Mixture,0.25 μM Oligo d(T), 0.25 μM Random Primer, 40 U RNase inhibitor, and 125U MuLV reverse transcriptase (Applied Biosystems, Foster City, Calif.)for 10 minutes at 25° C., 30 minutes at 48° C., and 5 minutes at 95° C.To confirm the absence of DNA contamination, RNA samples on whichreverse transcriptase was not performed were also subjected to PCR. Theabsence of PCR product confirmed the lack of DNA contamination in theRNA samples.

Quantitative Real-time PCR: The SYBR Green Real-Time PCR Kit (AppliedBiosystems, Foster City, Calif.) was used to quantify cDNA expression ofAd5-hLMP-1(t)-GFP, alkaline phosphatase, osteocalcin, BMP-2, BMP-7 and18S (for normalization). 25 μL of reaction volume included 5 μL of cDNA,5 pmols of each primer, and 12.5 μL of 2×SYBR Green Master Mix (AppliedBiosystems, Foster City, Calif.). Primer sequences are listed inTable 1. Real-time PCR was performed with the following three-stepprotocol: step 1, 50° C. for 2 minutes; step 2, 95° C. for 10 minutes;and step 3, (95° C. for 15 seconds, 62° C. for 1 minute) times 40 cyclesusing the Gene Amp 5700 Sequence Detection system (Applied Biosystems,Foster City, Calif.). To confirm amplification specificity, the PCRproducts were subjected to a dissociation curve analysis. Thresholdcycles (Ct) of each reaction were normalized to those obtained for 18SRNA using the comparative -^(ΔΔ)Ct method, as described previously. AllPCR reactions were performed in triplicate.

Statistical Analysis—

Two tailed student t test was used to compare the treated group with thecontrol. A p value less than 0.05 was used to define statisticalsignificance.

Example 9

Calvarial Cell Culture—Rat calvarial cells, also known as ratosteoblasts (“ROB”), were obtained from 20-day pre-parturition rats aspreviously described. Boden et al., Endocrinology, 137(8):3401-07(1996). Primary cultures were grown to confluence (7 days), trypsinized,and passed into 6-well plates (1×10⁵ cells/35 mm well) as firstsubculture cells. The subculture cells, which were confluent at day 0,were grown for an additional 7 days. Beginning on day 0, media werechanged and treatments (Trm and/or BMPs) were applied, under a laminarflow hood, every 3 or 4 days. The standard culture protocol was asfollows: days 1-7, MEM, 10% FBS, 50 μg/ml ascorbic acid, ±stimulus; days8-14, BGJb medium, 10% FBS, 5 mM β-GlyP (as a source of inorganicphosphate to permit mineralization). Endpoint analysis of bone noduleformation and osteocalcin secretion was performed at day 14. The dose ofBMP was chosen as 50 ng/ml based on pilot experiments in this systemthat demonstrated a mid-range effect on the dose-response curve for allBMPs studied.

Example 10

Antisense Treatment and Cell Culture—To explore the potential functionalrole of LMP-1 during membranous bone formation, we synthesized anantisense oligonucleotide to block LMP-1 mRNA translation and treatedsecondary osteoblast cultures that were undergoing differentiationinitiated by glucocorticoid. Inhibition of RLMP expression wasaccomplished with a highly specific antisense oligonucleotide (having nosignificant homologies to known rat sequences) corresponding to a 25 bpsequence spanning the putative translational start site (SEQ ID NO: 35).Control cultures either did not receive oligonucleotide or they receivedsense oligonucleotide. Experiments were performed in the presence(preincubation) and absence of lipofectamine. Briefly, 22 μg of sense orantisense RLMP oligonucleotide was incubated in MEM for 45 minutes atroom temperature. Following that incubation, either more MEM orpre-incubated lipofectamine/MEM (7% v/v; incubated 45 minutes at roomtemperature) was added to achieve an oligonucleotide concentration of0.2 μM. The resulting mixture was incubated for 15 minutes at roomtemperature. Oligonucleotide mixtures were then mixed with theappropriate medium, that is, MEM/Ascorbate/±Trm, to achieve a finaloligonucleotide concentration of 0.1 μM.

Cells were incubated with the appropriate medium (±stimulus) in thepresence or absence of the appropriate oligonucleotides. Culturesoriginally incubated with lipofectamine were re-fed after 4 hours ofincubation (37° C.; 5% CO₂) with media containing neither lipofectaminenor oligonucleotide. All cultures, especially cultures receivingoligonucleotide, were refed every 24 hours to maintain oligonucleotidelevels.

LMP-1 antisense oligonucleotide inhibited mineralized nodule formationand osteocalcin secretion in a dose-dependent manner, similar to theeffect of BMP-6 oligonucleotide. The LMP-1 antisense block in osteoblastdifferentiation could not be rescued by addition of exogenous BMP-6,while the BMP-G antisense oligonucleotide inhibition was reversed withaddition of BMP-6. This experiment further confirmed the upstreamposition of LMP-1 relative to BMP-6 in the osteoblast differentiationpathway. LMP-1 antisense oligonucleotide also inhibited spontaneousosteoblast differentiation in primary rat osteoblast cultures.

Example 11 Quantitation of Mineralized Bone Nodule Formation

Cultures of ROBs prepared according to Examples 9 and 10 were fixedovernight in 70% ethanol and stained with von Kossa silver stain. Asemi-automated computerized video image analysis system was used toquantitate nodule count and nodule area in each well. Boden et al.,Endocrinology, 137(8):3401-07 (1996). These values were then divided tocalculate the area per nodule values. This automated process wasvalidated against a manual counting technique and demonstrated acorrelation coefficient of 0.92 (p<0.000001). All data are expressed asthe mean ±standard error of the mean (S.E.M.) calculated from 5 or 6wells at each condition. Each experiment was confirmed at least twiceusing cells from different calvarial preparations.

Example 12

Quantitation of Osteocalcin Secretion—Osteocalcin levels in the culturemedia were measured using a competitive radioimmunoassay with amonospecific polyclonal antibody (Pab) raised in our laboratory againstthe C-terminal nonapeptide of rat osteocalcin as described in Nanes etal., Endocrinology, 127:588 (1990). Briefly, 1 μg of nonapeptide wasiodinated with 1 mCi ¹²⁵I—Na by the lactoperoxidase method. Tubescontaining 200 μl of assay buffer (0.02 M sodium phosphate, 1 mM EDTA,0.001% thimerosal, 0.025% BSA) received media taken from cell culturesor osteocalcin standards (0-12,000 fmole) at 100 μl/tube in assaybuffer. The Pab (1:40,000; 100 μl) was then added, followed by theiodinated peptide (12,000 cpm; 100 μl). Samples tested for non-specificbinding were prepared similarly but contained no antibody. Bound andfree PAbs were separated by the addition of 700 μl goat anti-rabbit IgG,followed by incubation for 18 hours at 4° C. After samples werecentrifuged at 1200 rpm for 45 minutes, the supernatants were decantedand the precipitates counted in a gamma counter. Osteocalcin values werereported in fmole/100 μl, which was then converted to pmole/ml medium(3-day production) by dividing those values by 100. Values wereexpressed as the mean ±S.E.M. of triplicate determinations for 5-6 wellsfor each condition. Each experiment was confirmed at least two timesusing cells from different calvarial preparations.

Example 13

Effect of Trm and RLMP on Mineralization In Vitro—There was littleapparent effect of either the sense or antisense oligonucleotides on theoverall production of bone nodules in the non-stimulated cell culturesystem. When ROBs were stimulated with Trm, however, the antisenseoligonucleotide to RLMP inhibited mineralization of nodules by >95%. Theaddition of exogenous BMP-6 to the oligonucleotide-treated cultures didnot rescue the mineralization of RLMP-antisense-treated nodules.

Osteocalcin has long been synonymous with bone mineralization, andosteocalcin levels have been correlated with nodule production andmineralization. The RLMP-antisense oligonucleotide significantlydecreases osteocalcin production, but the nodule count inantisense-treated cultures does not change significantly. In this case,the addition of exogenous BMP-6 only rescued the production ofosteocalcin in RLMP-antisense-treated cultures by 10-15%. This suggeststhat the action of RLMP is downstream of, and more specific than, BMP-6.

Example 14

Harvest and Purification of RNA—Cellular RNA from duplicate wells ofROBs (prepared according to Examples 9 and 10 in 6-well culture dishes)was harvested using 4M guanidine isothiocyanate (GIT) solution to yieldstatistical triplicates. Briefly, culture supernatant was aspirated fromthe wells, which were then overlayed with 0.6 ml of GIT solution perduplicate well harvest. After adding the GIT solution, the plates wereswirled for 5-10 seconds. Samples were saved at −70° C. for up to 7 daysbefore further processing.

RNA was purified by a slight modification of standard methods accordingto Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.,chapter 7.19, Cold Spring Harbor Press (1989). Briefly, thawed samplesreceived 60 μl 2.0 M sodium acetate (pH 4.0), 550 μl phenol (watersaturated) and 150 μl chloroform:isoamyl alcohol (49:1). Aftervortexing, the samples were centrifuged (10000×g; 20 minutes; 4° C.),the aqueous phase transferred to a fresh tube, 600 μl isopropanol wasadded and the RNA precipitated overnight at −20° C.

Following the overnight incubation, the samples were centrifuged(10000×g; 20 minutes) and the supernatant was aspirated gently. Thepellets were resuspended in 400 μl DEPC-treated water, extracted oncewith phenol:chloroform (1:1), extracted with chloroform:isoamyl alcohol(24:1) and precipitated overnight at −20° C. after addition of 40 μlsodium acetate (3.0 M; pH 5.2) and 1.0 ml absolute ethanol. To recoverthe cellular RNA, the samples were centrifuged (10000×g; 20 min), washedonce with 70% ethanol, air dried for 5-10 minutes and resuspended in 20μl of DEPC-treated water. RNA concentrations were calculated fromoptical densities that were determined with a spectrophotometer.

Example 15 Reverse Transcription-Polymerase Chain Reaction

Heated total RNA (5 μg in 10.5. μl total volume DEPC-H₂O at 65° C. for 5minutes) was added to tubes containing 4 μl 5×MMLV-RT buffer, 2 μldNTPs, 2 μl dT17 primer (10 pmol/ml), 0.5 μl RNAsin (40 U/ml) and 1 μlMMLV-RT (200 units/μl). The samples were incubated at 37° C. for 1 hour,then at 95° C. for 5 minutes to inactivate the MMLV-RT. The samples werediluted by addition of 80 μl of water.

Reverse-transcribed samples (5 μl) were subjected to polymerase-chainreaction using standard methodologies (50 μl total volume). Briefly,samples were added to tubes containing water and appropriate amounts ofPCR buffer, 25 mM MgCl₂, dNTPs, forward and reverse primers forglyceraldehyde 3-phosphate dehydrogenase (GAP, a housekeeping gene)and/or BMP-6), ³²P-dCTP, and Taq polymerase. Unless otherwise noted,primers were standardized to run consistently at 22 cycles (94° C., 30″;58° C., 30″; 72° C., 20″).

Example 16

Quantitation of RT-PCR Products by Polyacrylamide Gel Electrophoresis(PAGE) and Phosphorimager Analysis—RT-PCR products received 5 μl/tubeloading dye, were mixed, heated at 65° C. for 10 min and centrifuged.Ten μl of each reaction was subjected to PAGE (12% polyacrylamide:bis;15 V/well; constant current) under standard conditions. Gels were thenincubated in gel preserving buffer (10% v/v glycerol, 7% v/v aceticacid, 40% v/v methanol, 43% deionized water) for 30 minutes, dried (80°C.) in vacuo for 1-2 hours and developed with an electronically-enhancedphosphoresence imaging system for 6-24 hours. Visualized bands wereanalyzed. Counts per band were plotted graphically.

Example 17

Differential Display PCR—RNA was extracted from cells stimulated withglucocorticoid (Trm, 1 nM). Heated, DNase-treated total RNA (5 μg in10.5 μl total volume in DEPC-H₂O at 65° C. for 5 minutes) was reversetranscribed as described in Example 7, but H-T₁₁M (SEQ ID. NO: 4) wasused as the MMLV-RT primer. The resulting cDNAs were PCR-amplified asdescribed above, but with various commercial primer sets (for example,H-T₁₁G (SEQ ID NO: 4) and H-AP-10 (SEQ ID. NO: 5); GenHunter Corp,Nashville, Tenn.). Radiolabelled PCR products were fractionated by gelelectrophoresis on a DNA sequencing gel. After electrophoresis, theresulting gels were dried in vacuo and autoradiographs were exposedovernight. Bands representing differentially-expressed cDNAs wereexcised from the gel and reamplified by PCR using the method of Conneret al., Proc. Natl. Acad. Sci. USA, 88:278 (1983). The products of PCRreamplification were cloned into the vector PCR-II (TA cloning kit;InVitrogen, Carlsbad, Calif.).

Example 18

Screening of a UMR 106 Rat Osteosarcoma Cell cDNA Library—A UMR 106library (2.5×10¹⁰ pfu/ml) was plated at 5×10⁴ pfu/ml onto agar plates(LB bottom agar) and the plates were incubated overnight at 37° C.Filter membranes were overlaid onto plates for two minutes. Onceremoved, the filters were denatured, rinsed, dried and UV cross-linked.The filters were then incubated in pre-hybridization buffer (2×PIPES [pH6.5], 5% formamide, 1% SDS and 100 μg/ml denatured salmon sperm DNA) for2 h at 42° C. A 260 base-pair radiolabelled probe (SEQ ID NO: 3; ³²Plabeled by random priming) was added to the entire hybridizationmix/filters, followed by hybridization for 18 hours at 42° C. Themembranes were washed once at room temperature (10 min, 1×SSC, 0.1 SDS)and three times at 55° C. (15 min, 0.1×SSC, 0.1% SDS).

After they were washed, the membranes were analyzed by autoradiographyas described above. Positive clones were plaque purified. The procedurewas repeated with a second filter for four minutes to minimize spuriouspositives. Plaque-purified clones were rescued as lambda SK(−)phagemids. Cloned cDNAs were sequenced as described below.

Example 19

Sequencing of Clones—Cloned cDNA inserts were sequenced by standardmethods. Ausubel et al., Current Protocols in Molecular Biology, WileyInterscience (1988) Briefly, appropriate concentrations of terminationmixture, template and reaction mixture were subjected to an appropriatecycling protocol (95° C., 30 s; 68° C., 30 s; 72° C., 60 s; ×25). Stopmixture was added to terminate the sequencing reactions. After heatingat 92° C. for 3 minutes, the samples were loaded onto a denaturing 6%polyacrylamide sequencing gel (29:1 acrylamide:bis-acrylamide). Sampleswere electrophoresed for about 4 hours at 60 volts, constant current.After electrophoresis, the gels were dried in vacuo andautoradiographed.

The autoradiographs were analyzed manually. The resulting sequences werescreened against the databases maintained by the National Center forBiotechnology Information (NIH, Bethesda, Md.;http://www.ncbi.nim.nih.go-vl) using the BLASTn program set with defaultparameters. Based on the sequence data, new sequencing primers wereprepared and the process was repeated until the entire gene had beensequenced. All sequences were confirmed a minimum of three times in bothorientations.

Nucleotide and amino acid sequences were also analyzed using the PCGENEsoftware package (version 16.0). Percent homology values for nucleotidesequences were calculated by the program NALIGN, using the followingparameters: weight of non-matching nucleotides, 10; weight ofnon-matching gaps, 10; maximum number of nucleotides considered, 50; andminimum number of nucleotides considered, 50. For amino acid sequences,percent homology values were calculated using PALIGN. A value of 10 wasselected for both the open gap cost and the unit gap cost.

Example 20

Cloning of RLMP cDNA—The differential display PCR amplification productsdescribed in Example 17 contained a major band of approximately 260 basepairs. This sequence was used to screen a rat osteosarcoma (UMR 106)cDNA library. Positive clones were subjected to nested primer analysisto obtain the primer sequences necessary for amplifying the full lengthcDNA. (SEQ. ID NOs: 11, 12, 29, 30 and 31). One of those positive cloneswhich was selected for further study was designated clone 10-4.

Sequence analysis of the full-length cDNA in clone 10-4, determined bynested primer analysis, showed that clone 10-4 contained the original260 base-pair fragment identified by differential display PCR. Clone10-4 (1696 base pairs; SEQ ID NO: 2) contains an open reading frame of1371 base pairs encoding a protein having 457 amino acids (SEQ ID NO:1). The termination codon, TGA, occurs at nucleotides 1444-1446. Thepolyadenylation signal at nucleotides 1675-1680, and adjacent poly(A)⁺tail, was present in the 3′ noncoding region. There were two potentialN-glycosylation sites, Asn-Lys-Thr and Asn-Arg-Thr, at amino acidpositions 113-116 and 257-259 in SEQ ID NO: 1, respectively. Twopotential cAMP- and cGMP-dependent protein kinase phosphorylation sites,Ser and Thr, were found at amino acid positions 191 and 349,respectively. There were five potential protein kinase C phosphorylationsites, Ser or Thr, at amino acid positions 3, 115, 166, 219, 442. Onepotential ATP/GTP binding site motif A (P-loop),Gly-Gly-Ser-Asn-Asn-Gly-Lys-Thr, was determined at amino acid positions272-279.

In addition, two highly conserved putative LIM domains were found atamino acid positions 341-391 and 400-451. The putative LIM domains inthis newly identified rat cDNA clone showed considerable homology withthe LIM domains of other known LIM proteins. However, the overallhomology with other rat LIM proteins was less than 25%. RLMP (alsodesignated 10-4) has 78.5% amino acid homology to the human enigmaprotein (see U.S. Pat. No. 5,504,192), but only 24.5% and 22.7% aminoacid homology to its closest rat homologs, CLP-36 and RIT-18,respectively.

Example 21

Northern Blot Analysis of RLMP Expression—Thirty μg of total RNA fromROBs, prepared according to Examples 17 and 18, was size fractionated byformaldehyde gel electrophoresis in 1% agarose flatbed gels andosmotically transblotted to nylon membranes. The blot was probed with a600 base pair EcoR1 fragment of full-length 10-4 cDNA labeled with³²P-dCTP by random priming.

Northern blot analysis showed a 1.7 kb mRNA species that hybridized withthe RLMP probe. RLMP mRNA was up-regulated approximately 3.7-fold inROBs after 24 hours exposure to BMP-6. No up-regulation of RMLPexpression was seen in BMP-2 or BMP4-stimulated ROBs at 24 hours

Example 22

Statistical Methods—For each reported nodule/osteocalcin result, datafrom 5-6 wells from a representative experiment were used to calculatethe mean ±S.E.M. Graphs may be shown with data normalized to the maximumvalue for each parameter to allow simultaneous graphing of nodulecounts, mineralized areas and osteocalcin.

For each reported RT-PCR, RNase protection assay or Western blotanalysis, data from triplicate samples of representative experiments,were used to determine the mean ±S.E.M. Graphs may be shown normalizedto either day 0 or negative controls and expressed as fold-increaseabove control values. Statistical significance was evaluated using aone-way analysis of variance with post-hoc multiple comparisoncorrections of Bonferroni as appropriate. D. V. Huntsberger, “TheAnalysis of Variance,” in Elements of Statistical Variance, P.Billingsley (ed.), pp. 298-330, Allyn & Bacon Inc., Boston, Mass. (1977)and Sigmastat, Jandel Scientific, Corte Madera, Calif. Alpha levels forsignificance were defined as p<0.05.

Example 23

Detection of Rat LIM Mineralization Protein by Western BlotAnalysis—Polyclonal antibodies were prepared according to the methods ofEngland et al., Biochim. Biophys. Acta, 623:171 (1980) and Timmer etal., J. Biol. Chem., 268:24863 (1993).

HeLa cells were transfected with pCMV2/RLMP. Protein was harvested fromthe transfected cells according to the method of Hair et al., LeukemiaResearch, 20:1 (1996). Western Blot Analysis of native RLMP wasperformed as described by Towbin et al., Proc. Natl. Acad. Sci. USA,76:4350 (1979).

Example 24

Synthesis of the Rat LIMP-Unique (RLMPU)-Derived Human PCR Product—Basedon the sequence of the rat LMP-1 cDNA, forward and reverse PCR primers(SEQ ID NOs: 15 and 16) were synthesized and a unique 223 base-pairsequence was PCR amplified from the rat LMP-1 cDNA. A similar PCRproduct was isolated from human MG63 osteosarcoma cell cDNA with thesame PCR primers.

RNA was harvested from MG63 osteosarcoma cells grown in T-75 flasks.Culture supernatant was removed by aspiration and the flasks wereoverlayed with 3.0 ml of GIT solution per duplicate, swirled for 5-10seconds, and the resulting solution was transferred to 1.5 ml eppendorftubes (5 tubes with 0.6 ml/tube). RNA was purified by a slightmodification of standard methods, for example, see Sambrook et al.,Molecular Cloning: A Laboratory Manual, chapter 7, page 19, Cold SpringHarbor Laboratory Press (1989) and Boden 6t al., Endocrinology,138:2820-28 (1997). Briefly, the 0.6 ml samples received 60 μl 2.0 Msodium acetate (pH 4.0), 550 μl water saturated phenol and 150 μlchloroform:isoamyl alcohol (49:1). After addition of those reagents, thesamples were vortexed, centrifuged (10000×g; 20 min; 4° C.) and theaqueous phase transferred to a fresh tube. Isopropanol (600 μl) wasadded and the RNA was precipitated overnight at −20° C. The samples werecentrifuged (10000×g; 20 minutes) and the supernatant was aspiratedgently. The pellets were resuspended in 400 μl of DEPC-treated water,extracted once with phenol-chloroform (1:1), extracted withchloroform:isoamyl alcohol (24:1) and precipitated overnight at −20° C.in 40 μl sodium acetate (3.0 M; pH 5.2) and 1.0 ml absolute ethanol;After precipitation, the samples were centrifuged (10000×g; 20 min),washed once with 70% ethanol, air dried for 5-10 minutes and resuspendedin 20 μl of DEPC-treated water. RNA concentrations were derived fromoptical densities.

Total RNA (5 μg in 10.5 μl total volume in DEPC-H₂O) was heated at 65°C. for 5 minutes, and then added to tubes containing 4 μl 5.times.MMLV-RT buffer, 2 μl dNTPs, 2 μl dT17 primer (10 pmol/ml), 0.5 μl RNAsin(40 U/ml) and 1 μl MMLV-RT (200 units/μl). The reactions were incubatedat 37° C. for 1 hour. Afterward, the MMLV-RT was inactivated by heatingat 95° C. for 5 minutes. The samples were diluted by addition of 80 μLwater.

Transcribed samples (5 μl) were subjected to polymerase-chain reactionusing standard methodologies (50 μl total volume). Boden et al.,Endocrinology, 138:2820-28 (1997); Ausubel et al., “Quantitation of rareDNAs by the polymerase chain reaction”, in Current Protocols inMolecular Biology, chapter 15.31-1, Wiley & Sons, Trenton, N.J. (1990).Briefly, samples were added to tubes containing water and appropriateamounts of PCR buffer (25 mM MgCl₂, dNTPs, forward and reverse primers(for RLMPU; SEQ ID NOs: 15 and 16), ³²P-dCTP, and DNA polymerase.Primers were designed to run consistently at 22 cycles for radioactiveband detection and 33 cycles for amplification of PCR product for use asa screening probe (94° C., 30 sec, 58° C., 30 sec; 72° C., 20 sec).Sequencing of the agarose gel-purified MG63 osteosarcoma-derived PCRproduct gave a sequence more than 95% homologous to the RLMPU PCRproduct. That sequence is designated HLMP unique region (HLMPU; SEQ IDNO: 6).

Example 25

Screening of Reverse-Transcriptase-Derived MG63 CDNA—Screening wasperformed with PCR using specific primers (SEQ ID NOS: 16 and 17) asdescribed in Example 15. A 717 base-pair MG63 PCR product was agarosegel purified and sequenced with the given primers (SEQ. ID NOs: 12, 15,16, 17, 18, 27 and 28). Sequences were confirmed by a minimum of twotimes in both directions. The MG63 sequences were aligned against eachother and then against the full-length rat LMP cDNA sequence to obtain apartial human LMP cDNA sequence (SEQ ID NO: 7).

Example 26

Screening of a human Heart cDNA Library—Based on Northern blotexperiments, it was determined that LMP-1 is expressed at differentlevels by several different tissues, including human heart muscle. Ahuman heart cDNA library was therefore examined. The library was platedat 5×10⁴ pfu/ml onto agar plates (LB bottom agar) and plates were grownovernight at 37° C. Filter membranes were overlaid onto the plates fortwo minutes. Afterward, the filters denatured, rinsed, dried, UVcross-linked and incubated in pre-hybridization buffer (2×PIPES [pH6.5]; 5% formamide, 1% SDS, 100 g/ml denatured salmon sperm DNA) for 2 hat 42° C. A radiolabelled, LMP-unique, 223 base-pair probe (³²P, randomprimer labeling; SEQ ID NO: 6) was added and hybridized for 18 h at 42°C. Following hybridization, the membranes were washed once at roomtemperature (10 min, 1×SSC, 0.1% SDS) and three times at 55° C. (15 min,0.1×SSC, 0.1% SDS). Double-positive plaque-purified heart libraryclones, identified by autoradiography, were rescued as lambda phagemidsaccording to the manufacturers' protocols (Stratagene, La Jolla,Calif.).

Restriction digests of positive clones yielded cDNA inserts of varyingsizes. Inserts greater than 600 base-pairs in length were selected forinitial screening by sequencing. Those inserts were sequenced bystandard methods as described in Example 19.

One clone, number 7, was also subjected to automated sequence analysisusing primers corresponding to SEQ ID NOs: 11-14, 16 and 27. Thesequences obtained by these methods were routinely 97-100% homologous.Clone 7 (Partial Human LMP-1 cDNA from a heart library; SEQ. ID NO: 8)contained sequence that was more than 87% homologous to the rat LMP cDNAsequence in the translated region.

Example 27 Determination of Full-Length Human LMP-1 cDNA

Overlapping regions of the MG63 human osteosarcoma cell cDNA sequenceand the human heart cDNA clone 7 sequence were used to align those twosequences and derive a complete human cDNA sequence of 1644 base-pairs.NALIGN, a program in the PCGENE software package, was used to align thetwo sequences. The overlapping regions of the two sequences constitutedapproximately 360 base-pairs having complete homology except for asingle nucleotide substitution at nucleotide 672 in the MG63 cDNA (SEQID NO: 7) with clone 7 having an “A” instead of a EGO at thecorresponding nucleotide 516 (SEQ ID NO: 8).

The two aligned sequences were joined using SEQIN, another subprogram ofPCGENE, using the “G” substitution of the MG63 osteosarcoma cDNA clone.The resulting sequence is shown in SEQ ID NO: 9. Alignment of the novelhuman-derived sequence with the rat LMP-1 cDNA was accomplished withNALIGN. The full-length human LMP-1 cDNA sequence (SEQ. ID NO: 9) is87.3% homologous to the translated portion of rat LMP-1 cDNA sequence.

Example 28

Determination of Amino Acid Sequence of Human LMP1—The putative aminoacid sequence of human LMP-1 was determined with the PCGENE subprogramTRANSL. The open reading frame in SEQ ID NO: 9 encodes a proteincomprising 457 amino acids (SEQ. ID NO: 10). Using the PCGENE subprogramPalign, the human LMP-1 amino acid sequence was found to be 94.1%homologous to the rat LMP-1 amino acid sequence.

Example 29

Determination of the 5 Prime Untranslated Region of the Human LMPcDNA—MG63 5′ cDNA was amplified by nested RT-PCR of MG63 total RNA usinga 5′ rapid amplification of cDNA ends (5′ RACE) protocol. This methodincluded first strand cDNA synthesis using a lock-docking oligo (dT)primer with two degenerate nucleotide positions at the 31 end (Chenchiket al., CLONTECHniques. x :5 (1995); Borson et al., PC Methods Applic.,2:144 (1993)). Second-strand synthesis is performed according to themethod of Gubler et al., Gene. 25:263 (1983), with a cocktail ofEscherichia coli DNA polymerase I, RNase H, and E. coli DNA ligase.After creation of blunt ends with T4 DNA polymerase, double-strandedcDNA was ligated to the fragment(5′-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3′) (SEQ. ID NO: 19).Prior to RACE, the adaptor-ligated cDNA was diluted to a concentrationsuitable for Marathon RACE reactions (1:50). Adaptor-ligateddouble-stranded cDNA was then ready to be specifically cloned.

First-round PCR was performed with the adaptor-specific oligonucleotide,5′-CCATCCTAATACGACTCACTATAGGGC-3′ (AP1) (SEQ. ID NO: 20) as sense primerand a Gene Specific Primer (GSP) from the unique region described inExample 16 (HLMPU). The second round of PCR was performed using a nestedprimers GSP1-HLMPU (antisense/reverse primer) (SEQ. ID NO: 23) andGSP2-HLMPUF (SEQ. ID NO: 24) (see Example 16; sense/forward primer). PCRwas performed using a commercial kit (Advantage cDNA PCR core kit;CloneTech Laboratories Inc., Palo Alto, Calif.) that utilizes anantibody-mediated, but otherwise standard, hot-start protocol. PCRconditions for MG63 cDNA included an initial hot-start denaturation (94°C., 60 sec) followed by: 94° C., 30 sec; 60° C., 30 sec; 68° C., 4 min;30 cycles. The first-round PCR product was approximately 750 base-pairsin length whereas the nested PCR product was approximately 230base-pairs. The first-round PCR product was cloned into linearized pCR2.1 vector (3.9 Kb). The inserts were sequenced in both directions usingM13 Forward and Reverse primers (SEQ. ID NO: 11; SEQ. ID NO: 12).

Example 30

Determination of Full Length Human LMP-1 cDNA with 5 PrimeUTR—Overlapping MG63 human osteosarcoma cell cDNA 5′-UTR sequence (SEQID NO: 21), MG63 717 base-pair sequence (Example 17; SEQ ID NO: 8) andhuman heart cDNA clone 7 sequence (Example 26) were aligned to derive anovel human cDNA sequence of 1704 base-pairs (SEQ. ID NO: 22). Thealignment was accomplished with NALIGN, (both PCGENE and Omiga 1.0;Intelligenetics). Over-lapping sequences constituted nearly the entire717 base-pair region (Example 17) with 100% homology. Joining of thealigned sequences was accomplished with SEQIN.

Example 31

Construction of LIM Protein Expression Vector—The construction ofpHIS-5ATG LMP-1s expression vector was carried out with the sequencesdescribed in Examples 25 and 26. The 717 base-pair clone (Example 25;SEQ ID NO: 7) was digested with ClaI and EcoRV. A small fragment (about250 base-pairs) was gel purified. Clone 7 (Example 26; SEQ ID NO: 8) wasdigested with ClaI and xbaI and a 1400 base-pair fragment was gelpurified. The isolated 250 base-pair and 1400 base-pair restrictionfragments were ligated to form a fragment of about 1650 base-pairs.

Due to the single nucleotide substitution in Clone 7 (relative to the717 base-pair PCR sequence and the original rat sequence) a stop codonat translated base-pair 672 resulted. Because of this stop codon, atruncated (short) protein was encoded, hence the name LMP-1s. This wasthe construct used in the expression vector (SEQ ID NO: 32). The fulllength cDNA sequence with 5′ UTR (SEQ ID NO: 33) was created byalignment of SEQ ID NO: 32 with the 5′ RACE sequence (SEQ ID NO: 21).The amino acid sequence of LMP-1s (SEQ ID NO: 34) was then deduced as a223 amino acid protein and confirmed by Western blot (as in Example 23)to run at the predicted molecular weight of about 23.7 kD. The pHis-ATGvector (InVitrogen, Carlsbad, Calif.) was digested with EcoRV and XbaI.The vector was recovered and the 1650 base-pair restriction fragment wasthen ligated into the linearized pHis-ATG. The ligated product wascloned and amplified. The pHis-ATG-LMP-1s Expression vector, alsodesignated pHIS-A with insert HLMP-1s, was purified by standard methods.

Example 32

Induction of Bone Nodule Formation and Mineralization In Vitro with LMPExpression Vector—Rat Calvarial cells were isolated and grown insecondary culture according to Example 9. Cultures were eitherunstimulated or stimulated with glucocorticoid (CC) as described inExample 9. A modification of the Superfect Reagent (Qiagen, Valencia,Calif.) transfection protocol was used to transfect 3 μg/well of eachvector into secondary rat calvarial osteoblast cultures according toExample 25. Mineralized nodules were visualized by Von Kossa staining,as described in Example 11.

Human LMP-1s gene product overexpression alone induced bone noduleformation (about 203 nodules/well) in vitro. Levels of nodules wereapproximately 50% of those induced by the GC positive control (about 412nodules/well). Other positive controls included the pHisA-LMP-Ratexpression vector (about 152 nodules/well) and the pCMV2/LMP-Rat-FwdExpression vector (about 206-nodules/well), whereas the negativecontrols included the pCMV2/LMP-Rat-Rev. Expression vector (about 2nodules/well) and untreated (NT) plates (about 4 nodules/well). Thesedata demonstrate that the human cDNA was at least as osteoinductive asthe rat cDNA. The effect was less than that observed with GCstimulation, most likely due to suboptimal doses of Expression vector.

Example 33

LMP Induced Cell Differentiation In Vitro and In Vivo—The rat LMP cDNAin clone 10-4 (see Example 20) was excised from the vector bydouble-digesting the clone with NotI and ApaI overnight at 37° C. VectorpCMV2 MCS (InVitrogen, Carlsbad, Calif.) was digested with the samerestriction enzymes. Both the linear cDNA fragment from clone 10-4 andpCMV2 were gel purified, extracted and ligated with T4 ligase. Theligated DNA was gel purified, extracted and used to transform E. coliJM109 cells for amplification. Positive agar-colonies were picked,digested with NotI and ApaI and the restriction digests were examined bygel electrophoresis. Stock cultures were prepared of positive clones.

A reverse vector was prepared in analogous fashion except that therestriction enzymes used were XbaI and HindIII. Because theserestriction enzymes were used, the LMP cDNA fragment from clone 10-4 wasinserted into pRc/CMV2 in the reverse (that is, non-translatable)orientation. The recombinant vector produced is designated pCMV2/RLMP.

An appropriate volume of pCMV10-4 (60 nM final concentration is optimal(3 μg]; for this experiment a range of 0-600 nM/well [0-30 μg/well]final concentration is preferred) was resuspended in Minimal Eagle Media(MEM) to 450 μl final volume and vortexed for 10 seconds. Superfect wasadded (7.5 μl/ml final solution), the solution was vortexed for 10seconds and then incubated at room temperature for 10 minutes. Followingthis incubation, MEM supplemented with 10% FBS (1 ml/well; 6 ml/plate)was added and mixed by pipetting.

The resulting solution was then promptly pipetted (1 ml/well) ontowashed ROB cultures. The cultures were incubated for 2 hours at 37° C.in a humidified atmosphere containing 5% CO₂. Afterward, the cells weregently washed once with sterile PBS and the appropriate normalincubation medium was added.

Results demonstrated significant bone nodule formation in all rat cellcultures which were induced with pCMV10-4. For example, pCMV10-4transfected cells produced 429 nodules/well. Positive control cultures,which were exposed to Trm, produced 460 nodules/well. In contrast,negative controls, which received no treatment, produced 1 nodule/well.Similarly, when cultures were transfected with pCMV10-4 (reverse), nonodules were observed.

For demonstrating de novo bone formation in vivo, marrow was aspiratedfrom the hindlimbs of 4-5 week old normal rats (mu/+; heterozygous forrecessive athymic condition). The aspirated marrow cells were washed inalpha MEM, centrifuged, and RBCs were lysed by resuspending the pelletin 0.83% NH₄Cl in 10 mM Tris (pH 7.4). The remaining marrow cells werewashed 3× with MEM and transfected for 2 hours with 9 μg of pCMV-LMP-1s(forward or reverse orientation) per 3×10⁶ cells. The transfected cellswere then washed 2× with MEM and resuspended at a concentration of 3×10⁷cells/ml.

The cell suspension (100 μl) was applied via sterile pipette to asterile 2×5 mm type I bovine collagen disc (Sulzer Orthopedics, WheatRidge, Co.). The discs were surgically implanted subcutaneously on theskull, chest, abdomen or dorsal spine of 4-5 week old athymic rats(mu/mu). The animals were scarified at 3-4 weeks, at which time thediscs or surgical areas were excised and fixed in 70% ethanol. The fixedspecimens were analyzed by radiography and undecalcified histologicalexamination was performed on 5 μm thick sections stained with GoldnerTrichrome. Experiments were also performed using devitalized (guanidineextracted) demineralized bone matrix (Osteotech, Shrewsbury, N.J.) inplace of collagen discs.

Radiography revealed a high level of mineralized bone formation thatconformed to the form of the original collagen disc containing LMP-istransfected marrow cells. No mineralized bone formation was observed inthe negative control (cells transfected with a reverse-oriented versionof the LMP-1s cDNA that did not code for a translated protein), andabsorption of the carrier appeared to be well underway.

Histology revealed new bone trabeculae lined with osteoblasts in theLMP-1s transfected implants. No bone was seen along With partialresorption of the carrier in the negative controls.

Radiography of a further experiment in which 18 sets (9 negative controlpCMV-LMP-REV & 9 experimental pCMV-LMP-1s) of implants were added tosites alternating between lumbar and thoracic spine in athymic ratsdemonstrated 0/9 negative control implants exhibiting bone formation(spine fusion) between vertebrae. All nine of the pCMV-LMP-1s treatedimplants exhibited solid bone fusions between vertebrae.

Example 34

The Synthesis of pHIS-5′ ATG LMP-1s Expression Vector from the SequencesDemonstrated in Examples 10 and 11—The 717 base-pair clone (Example 25)was digested with ClaI and EcoRV (New England Biologicals, city, Mass.).A small fragment (about 250 base-pairs) was gel purified. Clone No. 7(Example 18) was digested with ClaI and XbaI. A 1400 base-pair fragmentwas gel purified from that digest. The isolated 250 base-pair and 1400base-pair cDNA fragments were ligated by standard methods to form afragment of about 1650 bp. The pHis-A vector (InVitrogen) was digestedwith EcoRV and XbaI. The linearized vector was recovered and ligated tothe chimeric 1650 base-pair cDNA fragment. The ligated product wascloned and amplified by standard methods, and the pHis-A-5′ ATG LMP-1sexpression vector, also denominated as the vector pHis-A with insertHLMP-1s, was deposited at the ATCC as previously described.

Example 35

The Induction of Bone Nodule Formation and Mineralization In Vitro WithpHis-5′ ATG LMP-1s Expression Vector—Rat calvarial cells were isolatedand grown in secondary culture according to Example 9. Cultures wereeither unstimulated or stimulated with glucocorticoid (GC) according toExample 9. The cultures were transfected with 3 μg of recombinant pHis-Avector DNA/well as described in Example 33. Mineralized nodules werevisualized by Von Kossa staining according to Example 12.

Human LMP-1s gene product overexpression alone (i.e., without GCstimulation) induced significant bone nodule formation (about 203nodules/well) in vitro. This is approximately 50% of the amount ofnodules produced by cells exposed to the GC positive control (about 412nodules/well). Similar results were obtained with cultures transfectedwith pHisA-LMP-Rat Expression vector (about 152 nodules/well) andpCMV2/LMP-Rat-Fwd (about 206 nodules/well). In contrast, the negativecontrol pCMV2/LMP-Rat-Rev yielded (about 2 nodules/well), whileapproximately 4 nodules/well were seen in the untreated plates. Thesedata demonstrate that the human LMP-1 cDNA was at least asosteoinductive as the rat LMP-1 cDNA in this model system. The effect inthis experiment was less than that observed with GC stimulation; but insome the effect was comparable.

Example 36

LMP Induces Secretion of a Soluble Osteoinductive Factor—Overexpressionof RLMP-1 or HLMP-1s in rat calvarial osteoblast cultures as describedin Example 24 resulted in significantly greater nodule formation thanwas observed in the negative control. To study the mechanism of actionof LIM mineralization protein conditioned medium was harvested atdifferent time points, concentrated to 10×, sterile filtered, diluted toits original concentration in medium containing fresh serum, and appliedfor four days to untransfected cells.

Conditioned media harvested from cells transfected with RLMP-1 orHLMP-1s at day 4 was approximately as effective in inducing noduleformation as direct overexpression of RLMP-1 in transfected cells.Conditioned media from cells transfected with RLMP-1 or HLMP-1 in thereverse orientation had no apparent effect on nodule formation. Nor didconditioned media harvested from LMP-1 transfected cultures before day 4induce nodule formation. These data suggest that expression of LMP-1caused the synthesis and/or secretion of a soluble factor, which did notappear in culture medium in effective amounts until 4 days posttransfection.

Since overexpression of rLMP-1 resulted in the secretion of anosteoinductive factor into the medium, Western blot analysis was used todetermine if LMP-1 protein was present in the medium. The presence ofrLMP-1 protein was assessed using antibody specific for LMP-1 (QDPDEE)and detected by conventional means. LMP-1 protein was found only in thecell layer of the culture and not detected in the medium.

Partial purification of the osteoinductive soluble factor wasaccomplished by standard 25% and 100% ammonium sulfate cuts followed byDE-52 anion exchange batch chromatography (100 mM or 500 mM NaCl). Allactivity was observed in the high ammonium sulfate, high NaCl fractions.Such localization is consistent with the possibility of a single factorbeing responsible for conditioning the medium.

All cited publications are hereby incorporated by reference in theirentirety.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be appreciated by one skilled in the art from reading thisdisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

1. A method of switching a differentiation of a cell from anon-osteogenic lineage into an osteogenic lineage comprising introducinginto said cell an agent capable of disrupting binding between a Smurf1protein and a Smad1/5 protein, wherein the agent capable of disruptingbinding between Smurf1 and Smad1/5 is selected from: i) an amino acidsequence-comprising SEQ ID NO: 51; or ii) a nucleic acid sequenceencoding the amino acid sequence comprising SEQ ID NO:
 51. 2. The methodof claim 1, wherein the step of introducing the agent comprisesoverexpressing an amino acid sequence which is at least 70% identical toa LMP protein or a fragment thereof in said cell.
 3. The method of claim2, wherein said cell is a myoblast.
 4. The method of claim 2, whereinsaid cell is cultured.
 5. The method of claim 2, wherein a nucleic acidsequence encodes an amino acid sequence which is at least 70% identicalto the LMP protein or a fragment thereof is introduced into the cell. 6.The method of claim 5, wherein the nucleic acid sequence is includedwithin a vector.
 7. The method of claim 5, wherein the nucleic acidsequence encodes for an amino acid sequence comprising SEQ ID. NO. 47.8. The method of claim 5, wherein the nucleic acid sequence encodes foran amino acid sequence comprising SEQ. ID. NO.
 49. 9. The method ofclaim 5, wherein the nucleic acid sequence encodes for an amino acidsequence comprising SEQ. ID. NO. 50 or SEQ. ID. NO.
 51. 10. The methodof claim 5, wherein the fragment of the LMP-1 protein is capable ofbinding a WW2 domain of a Smurf1 protein.
 11. A method of treating abone void comprising: (a) obtaining at least one cell of non-osteogeniclineage; (b) introducing into the at least one cell an agent capable ofdisrupting binding between a Smurf1 protein and a Smad1/5 protein; (c)culturing the at least one cell for a time sufficient to direct the atleast one cell to osteogenic lineage; (d) introducing the at least onecell of osteogenic lineage into a patient having the bone void, whereinthe agent capable of disrupting binding between Smurf-1 and Smad 1/5 isselected from: i) an amino acid sequence comprising SEQ ID NO: 51; orii) a nucleic acid sequence encoding the amino acid sequence comprisingSEQ ID NO:
 51. 12. The method of claim 1, wherein said at least one cellof non-osteogenic lineage is a myoblast.
 13. The method of claim 11,wherein said at least one cell of non-osteogenic lineage is derived fromthe patient.
 14. The method of claim 11, wherein said one cell ofnon-osteogenic lineage is cultured on a substrate shapeable into a shapeof the bone void.
 15. The method of claim 15, wherein said substrate isa mesh or a cage.
 16. The method of claim 11, wherein the agentcomprises a nucleic acid sequence encoding an amino acid sequence whichis at least 70% identical to the LMP protein or a fragment thereof. 17.The method of claim 16, wherein the nucleic acid sequence is includedwithin a vector.
 18. The method of claim 16, wherein the nucleic acidsequence encodes for an amino acid sequence comprising SEQ ID. NO. 47.19. The method of claim 16, wherein the nucleic acid sequence encodesfor an amino acid sequence comprising SEQ. ID. NO
 49. 20. The method ofclaim 16, wherein the nucleic acid sequence encodes for an amino acidsequence comprising SEQ. ID. NO. 50 or SEQ. ID. NO.
 51. 21. The methodof claim 16, wherein the fragment of the LMP-1 protein is capable ofbinding a WW2 domain of a Smurf1 protein.
 22. A method of generating acell culture model system for assessing the intracellular signalingpathways of bone growth factors comprising the steps of: (a) culturingmyocytes in a suitable medium, (b) transducing or transfecting a cDNAmolecule encoding an amino acid sequence which is at least 70% identicalto a truncated human LMP protein in said myocytes, (c) expressing thebone growth factors in the myocytes, (d) allowing sufficient time for aninhibition of myotubes in said myocytes, and (e) inducing said myocytesto exhibit at least one osteoblastic phenotype.
 23. The method of claim22, further comprising the step of examining the mechanisms of LMPinduction of the osteoblast.
 24. The method of claim 22, wherein thegrowth factors are selected from the group consisting of TGF-β and BMPgrowth factors.
 25. The method of claim 22, wherein the BMP is selectedfrom the group consisting of BMP-2, BMP-5, and BMP-6.
 26. The method ofclaim 22, wherein the myocytes are C₂C₁₂ cells.
 27. The method of claim22, wherein the transduction is done using a vector selected from thegroup consisting of a plasmid and a virus.
 28. The method of claim 22,wherein the truncated human LMP protein is selected from the groupconsisting of LMP-1, hLMP-1, truncated hLMP-1, hLMP-2, and hLMP-3.
 29. Amethod of assessing the intracellular signaling of a bone growth factorin myocytes comprising the steps of: a) culturing myocytes in a suitablemedium, b) transducing or transfecting a cDNA molecule encoding an aminoacid sequence which is at least 70% identical to a truncated human LMPprotein in said myocytes, c) expressing the bone growth factors in themyocytes, d) allowing sufficient time for the inhibition of myotubes insaid myocytes, e) inducing said myocytes to exhibit at least oneosteoblastic phenotype.
 30. The method of claim 29, further comprisingthe step of examining the mechanisms of LMP induction of the osteoblast.31. The method of claims 29, wherein the growth factors are selectedfrom the group consisting of TGF-β and BMP growth factors.
 32. Themethod of claim 29, wherein the myocytes are mammalian C₂C₁₂ cells. 33.The method of claim 29, wherein the transduction is done using a vectorselected from the group consisting of a plasmid and a virus.
 34. Themethod of claim 29, wherein the truncated human LMP protein is selectedfrom the group consisting of LMP-1, hLMP-1, truncated hLMP-1, hLMP-2,and hLMP-3.
 35. A method of screening the activity of exogenous factorson the intracellular signaling pathways of bone growth factorscomprising the steps of: a) culturing myocytes in a suitable medium, b)transducing or transfecting a cDNA molecule encoding an amino acidsequence which is at least 70% identical to a truncated human LMPprotein in said myocytes, c) expressing the bone growth factors in themyocytes, d) allowing sufficient time for the inhibition of myotubes insaid myocytes, e) inducing said myocytes to exhibit at least oneosteoblastic phenotype, and f) examining the effects of the externalfactors on the mechanisms of LMP induction of said myocytes of step (e).36. The method of claims 35, wherein the growth factors are selectedfrom the group consisting of TGF-β and BMP growth factors.
 37. Themethod of claim 35, wherein the BMP growth factor is selected from thegroup consisting of BMP-2, BMP-5, and BMP-6.
 38. The method of claim 35,wherein the myocytes are mammalian C₂C₁₂ cells.
 39. The method of claim35, wherein the transduction is done using a vector selected from thegroup consisting of a plasmid and a virus.